Anti-tissue factor antibodies and compositions

ABSTRACT

Isolated anti-tissue factor antibodies, immunoglobulins, cleavage products and other specified portions and variants thereof having enhanced ADCC activity, as well as anti-tissue factor antibody compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and methods of making and using thereof. The antibodies of the invention bind human tissue factor and demonstrate enhanced ADCC activity. Accordingly, the antibodies can be used in a variety of methods for diagnosing, treating, and/or preventing diseases involving tissue factor, where enhanced ADCC activity is desirable such as cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This divisional application claims the benefit of nonprovisional patentapplication Ser. No. 11/010,797, filed on Dec. 13, 2004 now U.S. Pat.No. 7,605,235, which claims the benefit of nonprovisional patentapplication Ser. No. 10/855,664, filed on 27 May 2004 which claims thebenefit of provisional Ser. No. 60/475,174, filed on 30 May 2003, whichare hereby incorporated by reference herein.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.10/855,664, filed May 27, 2004, which claims the benefit of USprovisional application No. 60/475,174 filed May 30, 2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antibodies which bind to human tissuefactor, including specified portions or variants thereof. The antibodiesof the invention have the ability to interact with effector cells toactivate innate immunity in addition to their human tissue factorneutralizing activity and are thus particularly useful in methods fortreating tumor cells. The invention also relates to nucleic acidsencoding such anti-tissue factor antibodies, complementary nucleicacids, vectors, host cells, and methods of making and using thereof,including therapeutic formulations, administration and devices.

2. Related Art

Tissue Factor (TF)

The coagulation of blood involves a cascading series of reactionsleading to the formation of fibrin. The coagulation cascade consists oftwo overlapping pathways, both of which are required for hemostasis. Theintrinsic pathway comprises protein factors present in circulatingblood, while the extrinsic pathway requires tissue factor (TF), which isexpressed on the cell surface of a variety of tissues in response tovascular injury (Davie et al., 1991, Biochemistry 30:10363). Whenexposed to blood, TF sets in motion a potentially explosive cascade ofactivation steps that result in the formation of an insoluble fibrinclot.

TF has been investigated as a target for anticoagulant therapy. TF is asingle chain, 263 amino acid membrane glycoprotein that functions as areceptor for factor VII and VIIa and thereby initiates the extrinsicpathway of the coagulation cascade in response to vascular injury. TF isa transmembrane cell surface receptor which serves as the receptor aswell as the cofactor for factor VIIa, forming a proteolytically activeTF:VIIa complex on cell surfaces (Ruf et al, (1992) J. Biol. Chem.267:6375-6381). In addition to its role in maintaining hemostasis,excess TF has been implicated in pathogenic conditions. Specifically,the synthesis and cell surface expression of TF has been implicated invascular disease (Wilcox et al., 1989, Proc. Natl. Acad. Sci, 86:2839)and gram-negative septic shock (Warr et al., 1990, Blood 75:1481).

TF Antagonists

Various anti-TF antibodies are known. For example, Carson et al, (1987,Blood 70:490-493) discloses a hybridoma producing monoclonal antibodyprepared by immunizing mice with TF purified by affinity chromatographyon immobilized factor VII. Ruf et al, (1991, Thrombosis and Haemostasis66:529) characterized the anticoagulant potential of murine monoclonalantibodies against human TF. The ability of monoclonal antibodies thattarget the FVII binding site on TF, is dependent on their ability tocompete with FVII for binding to TF and formation of the TF/VIIacomplex, which is rapidly formed when TF contacts plasma. Suchantibodies were thus relatively slow inhibitors of TF in plasma. Onemonoclonal antibody, TF8-5G9, was capable of inhibiting the TF/VIIacomplex, thus providing an immediate anticoagulant effect in plasma.This antibody is disclosed in U.S. Pat. Nos. 6,001,978, 5,223,427, and5,110,730. Ruf et al, suggested (supra) that mechanisms that inactivatethe TF/VIIa complex, rather than prevent its formation, may providestrategies for interruption of coagulation in vivo. In contrast to otherantibodies that inhibit factor VII binding to TF, TF8-5G9 shows onlysubtle and indirect effects on factor VII or factor VIIa binding to thereceptor. TF8-5G9 binds to the extracellular domain of TF with ananomolar binding constant to block the formation of the TF:F.VIIa:F.Xternary initiation complex (Huang et al, J. Mol. Biol. 275:873-8941998).

Anti-TF monoclonal antibodies have been shown to inhibit TF activity invarious species (Morissey et al., 1988, Thromb. Res. 52:247-260) andneutralizing anti-TF antibodies have been shown to prevent death in ababoon model of sepsis (Taylor et al, Circ. Shock, 33:127 (1991)), andattenuate endotoxin induced DIC in rabbits (Warr et al, (1990) Blood75:1481).

W0 96/40921 discloses CDR-grafted anti-TF antibodies derived from theTF8-5G9 antibody. Other humanized or human anti-TF antibodies aredisclosed in Presta et al, Thromb Haemost 85:379-389 (2001), EP1069185,WO 01/70984 and WO03/029295.

The Role of TF in Cancer

Tissue factor (TF) is a cell surface receptor best known for its role ininitiating blood coagulation upon injury. Tissue factor is alsooverexpressed on a variety of malignant tumors and isolated human tumorcell lines, suggesting a role in tumor growth and survival. TF is notproduced by healthy endothelial cells lining normal blood vessels but isexpressed on these cells in tumor vessels. It appears to play a role inboth vasculogenesis, the formation of new blood vessels in thedeveloping animal and in angiogenesis, the sprouting of new capillariesfrom existing arteries, in normal and malignant adult tissues.

Aberrant expression of TF on endothelial and tumor cells in a variety ofbreast, colorectal, lung and pancreatic cancers has been linked to anincrease in tumor microvessel density and upregulated VEGF expression.Tumor cells over expressing TF are also thought to be responsible forthe thrombotic complications associated with cancer. Thus there is arationale for the inhibition of tissue factor in the treatment ofcancer.

WO94/05328 discloses the use of anti-TF antibodies to inhibit the onsetand progression of metastasis by abolishing the prolonged adherence ofmetastazing cells in the microvasculature thereby inhibiting metastasis,but does not disclose any effect on the growth of established tumorcells. Given the complexity in the factors regulating tumorvascularization as well as the incomplete understanding of the role oftissue factor as a receptor mediating cellular growth in both tumorgrowth and wound healing, it is possible that blockade of TF could playeither a critical or a redundant role in the pathogenesis of cancer orother diseases characterized by inappropriate angiogenic activity.Inhibition or targeting of TF may therefore be a useful anti-tumorstrategy that could affect the survival of TF overexpressing tumor cellsdirectly by inhibiting TF mediated cellular signaling or otheractivities. In addition, this approach may prevent tumor growthindirectly via an antiangiogenic mechanism by inhibiting the growth orfunction of TF expressing intra-tumoral endothelial cells.

TF and Angiogenesis

Angiogenesis is the process of generating new capillary blood vessels,and results from activated proliferation of endothelial cells.Neovascularization is tightly regulated, and occurs only duringembryonic development, tissue remodeling, wound healing and periodiccycle of corpus luteum development (Folkman and Cotran, Relation ofvascular proliferation to tumor growth, Int. Rev. Exp. Pathol.'16,207-248 (1976)).

There is now considerable evidence that tumor growth and cancerprogression requires angiogenesis and neovascularization, blood vesselgrowth and extension, in order to provide tumor tissue with nutrientsand oxygen, to carry away waste products and to act as conduits for themetastasis of tumor cells to distant sites (Folkman, et al. N Engl J Med285: 1181-1186, 1971 and Folkman, et al. N Engl J Med 333: 1757-1763,1995). Nevertheless, tissue and tumor angiogenesis andneovascularization represent complex processes mediated by the interplayof cellularly produced factors: including TNFalpha, VEGF, and tissuefactor. Studies show that the pathways leading to upregulation of VEGFand TF overlap (Chen J. et al. (2001) Thromb. Haemost. 86-334-5), twomajor players in the initiation of new blood vessel formation.

Endothelial cells normally proliferate much more slowly than other typesof cells in the body. However, if the proliferation rate of these cellsbecomes unregulated, pathological angiogenesis can result. Pathologicalangiogenesis is involved in many diseases. For example, cardiovasculardiseases such as angioma, angiofibroma, vascular deformity,atherosclerosis, synechia and edemic sclerosis; and opthalmologicaldiseases such as neovascularization after cornea implantation,neovascular glaucoma, diabetic retinopathy, angiogenic corneal disease,macular degeneration, pterygium, retinal degeneration, retrolentalfibroplasias, and granular conjunctivitis are related to angiogenesis.Chronic inflammatory diseases such as arthritis; dermatological diseasessuch as psoriasis, telangiectasis, pyogenic granuloma, seborrheicdermatitis, venous ulcers, acne, rosacea (acne rosacea or erythematosa),warts (verrucas), eczema, hemangiomas, lymphangiogenesis are alsoangiogenesis-dependent.

Vision can be impaired or lost because of various ocular diseases inwhich the vitreous humor is infiltrated by capillary blood. Diabeticretinopathy can take one of two forms, non-proliferative orproliferative. Proliferative retinopathy is characterized by abnormalnew vessel formation (neovascularization), which grows on the vitreoussurface or extends into the vitreous cavity. In advanced disease,neovascular membranes can occur, resulting in a traction retinaldetachment. Vitreous hemorrhages may result from neovascularization.Visual symptoms vary. A sudden severe loss of vision can occur whenthere is intravitreal hemorrhage. Visual prognosis with proliferativeretinopathy is more guarded if associated with severe retinal ischemia,extensive neovascularization, or extensive fibrous tissue formation.Macular degeneration, likewise takes two forms, dry and wet. Inexudative macular degeneration (wet form), which is much less common,there is formation of a subretinal network of choroidalneovascularization often associated with intraretinal hemorrhage,subretinal fluid, pigment epithelial detachment, and hyperpigmentation.Eventually, this complex contracts and leaves a distinct elevated scarat the posterior pole. Both forms of age-related macular degenerationare often bilateral and are preceded by drusen in the macular region.Another cause of loss of vision related to angiogenic etiologies aredamage to the iris. The two most common situations that result in theiris being pulled up into the angle are contraction of a membrane suchas in neovascular glaucoma in patients with diabetes or central retinalvein occlusion or inflammatory precipitates associated with uveitispulling the iris up into the angle (Ch. 99. The Merck Manual 17th Ed.1999).

Rheumatoid arthritis, an inflammatory disease, also results ininappropriate angiogenesis. The growth of vascular endothelial cells inthe synovial cavity is activated by the inflammatory cytokines, andresults in cartilage destruction and replacement with pannus in thearticulation (Koch A K, Polyerini P J and Leibovich S J. ArthritisRheum. 29, 471-479 (1986); Stupack D G, Storgard C M and Cheresh D A,Braz. J. Med. Biol. Res., 32, 578-581 (1999); Koch A K, Arthritis Rheum,41, 951 962 (1998)).

Psoriasis is caused by uncontrolled proliferation of skin cells. Fastgrowing cells require sufficient blood supply, and abnormal angiogenesisis induced in psoriasis (Folkman J., J. Invest. Dermatol., 59, 40-48(1972)).

Antibody Properties

IgG1 and IgG4 antibody isotypes differ in their ability to mediatecomplement dependent cytotoxicity (CDC) and antibody-dependent cellularcytotoxicity (ADCC). CDC is the lysing of a target in the presence ofcomplement. The complement activation pathway is initiated by thebinding of the first component of the complement system (C1q) to amolecule complexed with a cognate antigen. IgG1 is a strong inducer ofthe complement cascade and subsequent CDC activity, while IgG4 haslittle complement-inducing activity.

ADCC is a cell-mediated reaction in which nonspecific cytotoxic cellsthat express Fc receptors (FcRs) (e.g., Natural Killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. The IgG1 isotypesubclass binds with high affinity to the Fc receptor and contributes toADCC while IgG4 binds only weakly. The relative inability of IgG4 toactivate effector functions is drawback in those applications such asoncology where cell killing is a desirable characteristic of theantibody.

There remains a need in the art for variant structures of anti-TFantibodies with properties optimized for specific clinical indications.For example, optimizing ADCC and CDC antibody functions is generallydesirable for oncology indications. Other potential uses for anti-TFantibodies with enhanced ADCC activity include therapy for age relatedmacular degeneration or other angiogenesis related conditions in whichendothelial cells in aberrant blood vessels may express TF and can betargeted by ADCC. The inventors of this application have producedvariant anti-TF antibody structures designed to meet these needs.

SUMMARY OF THE INVENTION

The present invention provides isolated anti-tissue factor antibodies,immunoglobulins, cleavage products and other specified portions andvariants thereof having enhanced ADCC activity, as well as anti-tissuefactor antibody compositions, encoding or complementary nucleic acids,vectors, host cells, compositions, formulations, devices, transgenicanimals, transgenic plants, and methods of making and using thereof. Theantibodies of the invention bind human tissue factor and demonstrateenhanced ADCC activity. Accordingly, the antibodies can be used in avariety of methods for diagnosing, treating, and/or preventing diseasesinvolving tissue factor, where enhanced ADCC activity is desirable suchas cancer.

Thus, in one embodiment, the present invention provides at least oneisolated tissue factor antibody as described herein. In one embodiment,the antibody according to the present invention includes any protein orpeptide containing molecule that comprises at least a portion of acomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof derived from the antibody designatedTF8-5G9, in combination with a heavy chain or light chain variableregion, a framework region, and a heavy chain or light chain constantregion that is capable of interacting with effector cells or moleculesto activate innate immunity (e.g. complement lysis, NK cell killingoropsonization/phagocytosis by macrophages) and thus imparts ADCCactivity to the antibody, or any portion thereof, that can beincorporated into an antibody of the present invention. The antibodyCNTO 860 described herein is a human tissue factor antibody derived fromthe TF8-5G9 antibody in which the IgG4 Fc region was exchanged with anIgG1 Fc by recloning the humanized variable domain into a vectorcontaining the desired heavy chain constant regions; CH1, CH2 and CH3.The IgG1 antibody exhibits similar antigen binding and coagulationinhibiting properties as the IgG4 version, but demonstrates markedlyenhanced ADCC activity and is more potent at inhibiting the growth oforthotopic MDA-MB-231 human breast carcinoma xenografts.

Particular therapeutic antibodies of the invention include humanmonoclonal antibody CNTO 860, and functionally equivalent antibodieswhich have the human heavy chain and human light chain amino acidsequences as set forth in SEQ ID NO: 2 and SEQ ID NO: 4 respectively,and conservative modifications thereof. The antibody amino acid sequencecan further optionally comprise at least one specified substitution,insertion or deletion as described herein or as known in the art.

Other particular antibodies of the invention include human monoclonalantibodies which bind to an epitope defined by antibody CNTO 860, and/orwhich compete for binding to tissue factor with antibody CNTO 860, orwhich have other functional binding characteristics exhibited byantibody CNTO 860, and which compete for binding to Fc receptors withCNTO 860. Such antibodies include, for example, those which bind totissue factor with dissociation constant (KD) of 1-2 nanomolar or lessand which have an apparent binding affinity for FcγRII on U937 cells of40-100 nanomolar or less in a Fc receptor competition binding assay.

Isolated antibodies of the invention include those having antibodyisotypes with ADCC activity greater than exhibited by IgG4, such asIgG1, (e.g., IgG1κ and IgG1λ), IgG2, and IgG3, or hybrid isotypescontaining altered residues at specific residues in the Fc domains. Theantibodies can be full-length antibodies (e.g., IgG1) or can includeonly an antigen-binding portion and an Fc portion or domain capable ofeliciting effector functions including ADCC, complement activation, andC1q binding.

The present invention also provides at least one isolated human tissuefactor antibody as described herein, wherein:

-   -   the antibody competes with CNTO 860 for binding to human tissue        factor;    -   the antibody inhibits the TF:FVIa complex;    -   has an affinity to TF on MDA-MB-231 human breast carcinoma cells        as measured by flow cytometry equivalent to CNTO 860;    -   can block in vivo coagulation of human plasma in a prothrombin        assay at a concentration equivalent to CNTO 860; and    -   shows killing of MDA-MB-231 cells and HCT116 equivalent to CNTO        860 in a chromium release ADCC assay.

A tissue factor antibody can thus be screened for such correspondingactivities according to known methods.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding the specific tissue factor antibodies described herein. Suchnucleic acid molecules include those encoding all or a portion of amonoclonal tissue factor antibody as described herein, as well asrecombinant expression vectors which include such nucleic acids, andhost cells transfected with such vectors. Methods of producing theantibodies by culturing such host cells are also encompassed by theinvention. Particular nucleic acids provided by the invention comprisethe nucleotide sequences shown in SEQ ID NOs: 1 and SEQ ID NOs: 3, whichencode the heavy and light chains, respectively, of human tissue factorantibody CNTO 860. The present invention further provides recombinantvectors comprising said anti-tissue factor antibody nucleic acidmolecules, host cells containing such nucleic acids and/or recombinantvectors, as well as methods of making and/or using such antibody nucleicacids, vectors and/or host cells.

The present invention also provides at least one method for expressingat least one tissue factor antibody as described herein, in a host cell,comprising culturing a host cell as described herein under conditionswherein at least one tissue factor antibody is expressed in detectableand/or recoverable amounts.

The present invention also provides at least one composition comprising(a) an isolated anti-tissue factor antibody encoding nucleic acid and/orantibody as described herein; and (b) a suitable carrier or diluent. Thecarrier or diluent can optionally be pharmaceutically acceptable,according to known carriers or diluents. The composition can optionallyfurther comprise at least one further compound, protein or composition.

The present invention further provides at least one tissue factorantibody method or composition, for administering a therapeuticallyeffective amount to modulate or treat at least one tissue factor relatedcondition in a cell, tissue, organ, animal or patient and/or, prior to,subsequent to, or during a related condition, as known in the art and/oras described herein. The compositions include, for example,pharmaceutical and diagnostic compositions/kits, comprising apharmaceutically acceptable carrier and at least one tissue factorantibody. In one embodiment, the composition comprises a combination ofhuman antibodies or antigen-binding portions thereof, preferably each ofwhich binds to a distinct epitope. For example, a pharmaceuticalcomposition comprising a monoclonal antibody that mediates highlyeffective killing of target cells in the presence of effector cells canbe combined with a monoclonal antibody that inhibits tissue factoractivity and signaling in tumor angiogenesis, metastasis and growth.Thus, the combination provides multiple therapies tailored to providethe maximum therapeutic benefit. Compositions, e.g., pharmaceuticalcompositions, comprising a combination of at least one human tissuefactor-antibody, or antigen-binding portion thereof, and at least onebispecific or multispecific molecule of the invention, are also withinthe scope of the invention.

In yet another aspect of the invention, the tissue factor antibodies arederivatized, linked to or co-expressed with another functional molecule,e.g., another peptide or protein (e.g., an Fab′ fragment). For example,an antibody or antigen-binding portion of the invention can befunctionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody (e.g., to produce a bispecific or amultispecific antibody), a cytotoxin, a cellular ligand or an antigen.Accordingly, present invention encompasses a large variety of antibodyconjugates, bi- and multispecific molecules, and fusion proteins, all ofwhich bind to tissue factor expressing cells and which target othermolecules to the cells, or which bind to tissue factor and to othermolecules or cells.

Alternatively, antibodies of the invention can be co-administered withsuch therapeutic and cytotoxic agents, but not linked to them. They canbe coadministered simultaneously with such agents (e.g., in a singlecomposition or separately) or can be administered before or afteradministration of such agents. Such agents can include chemotherapeuticagents, such as dacarbazine, doxorubicin (adriamycin), cisplatin,bleomycin sulfate, carmustine, chlorambucil, cyclophosphamidehydroxyurea and combinations thereof. Antibodies of the invention alsocan be administered in conjunction with radiation therapy orhyperthermic therapy.

In yet another embodiment, the present invention provides a method forinhibiting the proliferation and/or growth of a cell expressing tissuefactor and inducing killing of a cell expressing tissue factor, bycontacting the cells with (e.g., administering to a subject) one or moreantibodies of the invention and/or related therapeutic compositions,derivatives etc. containing the antibodies as described above. In aparticular embodiment, the method comprises contacting cells expressingtissue factor either in vitro or in vivo with one or a combination oftissue factor antibodies of the invention in the presence of a humaneffector cell. The method can be employed in culture, e.g. in vitro orex vivo (e.g., cultures comprising cells expressing tissue factor andeffector cells). For example, a sample containing cells expressingtissue factor and effector cells can be cultured in vitro, and combinedwith an antibody of the invention.

Alternatively, the method can be performed in a subject, e.g., as partof an in vivo (e.g., therapeutic or prophylactic) protocol. For use inin vivo treatment and prevention of tissue factor mediated diseases,antibodies of the present invention are administered to patients (e.g.,human subjects) at therapeutically effective dosages (e.g., to inhibit,eliminate or prevent growth of cells expressing tissue factor or toinhibit angiogenesis and thus inhibit the growth of cells where growthis mediated by angiogenesis) using any suitable route of administrationfor antibody-based clinical products as are well known in the art, suchas by injection or infusion.

Accordingly, antibodies of the present invention can be used to treatand/or prevent a variety of tissue factor mediated diseases where ADCCactivity and other effector functions leading to increased killing andremoval of target cells is desirable by administering a suitable dosage(or series of dosages) of the antibodies to patients suffering from suchdiseases. Exemplary diseases that can be treated (e.g., ameliorated) orprevented using the methods and compositions of the invention include,but are not limited to, cancers, such as metastatic melanoma, prostatecancer, colon cancer, and renal carcinoma.

In a particular embodiment of the invention, the patient can beadditionally treated with a chemotherapeutic agent, radiation, or anagent that modulates, e.g., enhances, the expression or activity of anFc receptor, such as a cytokine. Typical cytokines for administrationduring treatment include granulocyte colony-stimulating fact or (G-CSF),granulocytemacrophage colony-stimulating factor (GM-C SF), interferon-y(IFN-y), and tumor necrosis factor (TNF). Typical therapeutic agentsinclude, among others, anti-neoplastic agents such as dacarbazine,doxorubicin (adriamycin), cisplatin, bleomycin sulfate, carmustine,chlorambucil, cyclophosphamide, and hydroxyurea.

As exemplified herein, tissue factor antibodies can be obtained directlyfrom hybridomas which express the antibody, or can be cloned andrecombinantly expressed in a host cell, such as a transfectoma (e.g., atransfectoma consisting of immortalized CHO cells or lymphocytic cells).Accordingly, the present invention provides methods for producingmonoclonal antibodies which bind to human tissue factor.

DESCRIPTION OF THE FIGURES

FIG. 1 is an alignment of the amino acid sequences of the mature heavychains of CNTO 859 and CNTO 860.

FIG. 2 shows diagrams of the expression vectors for CNTO860 heavy chain,p2401 (a) and light chain (b).

FIG. 3 is a graph showing the change in tumor volumes from eithercontrol animals, animals treated with either PBS or control human Ig andanimals treated with CNTO 859.

FIG. 4 is a bar graph representing the mean and standard deviation ofthe final tumor volumes from either control animals, animals treatedwith either PBS or control IgG and animals treated with CNTO 859.

FIG. 5 shows the tumor incidence rate in animals treated with eitherPBS, control Ig or CNTO 859 beginning on the same day as the tumor cellswere implanted.

FIG. 6 shows the tumor progression of MDA MB 231 xenografts as measuredby volume in animals treated with either PBS, control human Ig orvarious dosages of CNTO 859. CNTO 859 was able to inhibit tumor growthat all concentrations. Tumor inhibition ranged from 90% at 0.1 mg/kg(p=0.0012 and p=0.0106, respectively, Wilcoxon two-sample test usingt-distribution) to 95% at any concentration above that.

FIG. 7 is a scatter plot showing the distribution of final tumor volumesfrom animals treated with either PBS, control human Ig or variousdosages of CNTO 859 (0.1, 1, 5, 10 and 20 mg/kg).

FIG. 8 is graph of tumor volumes over time for an experiment using humanbreast cancer cells MDA MB 231 xenografts implanted in miceorthotopically (in mammary tissue) and where the mice were treated witheither PBS, control human Ig, CNTO 859 Ala/Ala or various dosages (0.01,0.1 and 1 mg/kg) of CNTO 859 and CNTO 860.

FIG. 9 shows means and standard deviation of four of the groups from thesame experiment shown in FIG. 8, showing only the controls and CNTO 859and CNTO 860 at 0.1 mg/kg.

FIG. 10 is a graphical representation of each of the individual finaltumor volumes and means from each group in the same experiment as FIG.8.

FIG. 11. shows the tumor incidence data from the same experiment as inFIG. 8.

FIG. 12 is a graph showing the binding data for CNTO859 and CNTO860 toMDA-231 cells.

FIG. 13 is a graph showing the relationship between antibodyconcentration and the inhibition of Fxa activation.

FIG. 14 is a graph showing the relationship between antibodyconcentration and prolongation of coagulation time.

FIG. 15 is a bar graph showing the relative abilities of CNTO859, theCNTO859 with ala/ala mutations in the hinge region, and CNTO860 comparedto non-specific and complete cell lysis by Triton-X.

FIG. 16 is a graph showing the 10- to 100-fold enhancement of Fcreceptor binding affinity of CNTO860 v CNTO859 in a competition assayusing radiolabeled control antibody bound to U937 cells.

FIG. 17 is graph showing the mean tumor volume over time for micebearing human breast tumor cells, MDA MB-231, implanted in the mammaryfat pad and treated with 0.1 mg/kg of F105, CNTO 859, CNTO 860, CNTO 859Ala/Ala and CNTO 860 Ala/Ala antibody or an equivalent volume of PBS.

FIG. 18 is a scatter plot showing the final tumor weights for the miceas described for FIG. 17.

FIG. 19 is a graph showing the percent of mice (8 per group) with orwithout measurable sized tumors over the course of the experiment bytreatment group as described for FIG. 17.

FIG. 20 is graph showing human breast tumor cell (MDA MB-231) growthover time in SCID beige mice treated with anti-human tissue factor (CNTO860) plus an anti-mouse tissue factor (PHD 126) both of which ofisotypes with capable of eliciting effector functions for the respectivespecies. F105 and vCam are isotype matched control antibodies. Antibodyinjections were given once per week for 8 weeks.

FIG. 21 is scatter plot of the individual final tumor masses for theexperiment as described for FIG. 20.

FIG. 22 is a plot of the percent of tumor free mice by group over timein the experiment as described for FIG. 20.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated, recombinant and/or syntheticanti-tissue factor monoclonal antibodies having enhanced ADCC activity,as well as compositions and encoding nucleic acid molecules comprisingat least one polynucleotide encoding such antibodies.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

The terms “tissue factor protein” and “mammalian tissue factor protein”are used to refer to a polypeptide having an amino acid sequencecorresponding to a naturally occurring mammalian tissue factor or arecombinant tissue factor as described below. Naturally occurring TFincludes human species as well as other animal species such as rabbit,rat, porcine, non human primate, equine, murine, and ovine tissue factor(see, for example, Hartzell et al., (1989) Mol. Cell. Biol.,9:2567-2573; Andrews et al., (1991) Gene, 98:265-269; and Takayenik etal., (1991) Biochem. Biophys. Res. Comm., 181:1145-1150). The amino acidsequence of human tissue factor is shown in FIG. 13 (SEQ ID NO: 13). Theamino acid sequence of the other mammalian tissue factor proteins aregenerally known or obtainable through conventional techniques.

A “TF mediated or associated process or event”, or equivalently, an“activity associated with TF”, according to the present invention is anyevent which is mediated by the presence of TF. A “TF related disease ordisorder” is meant to diseases or disorders which may be impactedthrough the inhibition of TF, particularly the inhibition of tumorgrowth on tissue factor expressing cells, but also includes other tissuefactor mediated diseases such as chronic thromboembolic diseases ordisorders associated with fibrin formation including vascular disorderssuch as deep venous thrombosis, arterial thrombosis, stroke, tumormetastasis, thrombolysis, arteriosclerosis and restenosis followingangioplasty, acute and chronic indications such as inflammation, septicshock, septicemia, hypotension, adult respiratory distress syndrome(ARDS), disseminated intravascular coagulopathy (DIC) and otherdiseases.

The term “ADCC activity” stands for antibody-dependent cell-mediatedcytotoxicity and means the phenomenon of antibody-mediated target celldestruction by non-sensitized effector cells. The identity of the targetcell varies, but it must have bound surface immunoglobulin G whose Fcportion is intact. The effector cell is a “killer” cell possessing Fcreceptors. It may be a lymphocyte lacking conventional B- or T-cellmarkers, or a monocyte, macrophage, or polynuclear leukocyte, dependingon the identity of the target cell. The reaction is complementindependent. The ADCC activity of an antibody of the present inventionis “enhanced”, if its ability to demonstrate ADCC mediated cell killingsurpasses the ability of an anti-TF IgG4 antibody, as determined in astandard in vivo or in vitro assay of cell killing, such as the assaysdiscussed herein. Preferably, the anti-TF with enhanced ADCC activityachieves the same effect (prevention or inhibition of tumor cell growth)at a lower dose and/or in a shorter time than a reference IgG4 antibody.Preferably, the difference between the potency of an antibody within thescope of the present invention and a reference antibody is at leastabout 1-fold, more preferably at least about 2-fold, even morepreferably at least about 3-fold, most preferably at least about 5-fold,as determined by side-by-side comparison in a selected standard chromiumrelease ADCC assay.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

“Effector functions” of antibodies or antibody analogs as it is usedherein are processes by which pathogens or abnormal cells, e.g. tumorcells, are destroyed and removed from the body. Innate and adaptiveimmune responses use most of the same effector mechanisms to eliminatepathogens including ADCC, CA (complement activation), C1q binding, andopsinization.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to conventional(polyclonal) antibody preparations which typically include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567) The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol.Biol. 222:581-597 (1991), for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567and Morrison et al., Proc. Nat. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable region(which are also known as the complementarity determining regions or CDR)residues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human 10 immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Reichmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

The term “recombinant antibody”, as used herein, includes all antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as (a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal for human immunoglobulin genes or ahybridoma prepared therefrom (described further in Section I, below),(b) antibodies isolated from a host cell transformed to express theantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Recombinant human antibodies have variable and constant regions derivedfrom human germline immunoglobulin sequences. In certain embodiments,however, such recombinant human antibodies can be subjected to in vitromutagenesis (or, when an animal transgenic for human Ig sequences isused, in vivo somatic mutagenesis) and thus the amino acid sequences ofthe VH and VL regions of the recombinant antibodies are sequences that,while derived from and related to human germline VH and VL sequences,may not naturally exist within the human antibody germline repertoire invivo.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to tissue factor is substantially free of antibodiesthat specifically bind antigens other than tissue factor). An isolatedantibody that specifically binds to an epitope, isoform or variant ofhuman tissue factor may, however, have cross-reactivity to other relatedantigens, e.g., from other species (e.g., tissue factor specieshomologs). Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals. In one embodiment of theinvention, a combination of “isolated” monoclonal antibodies havingdifferent specificities are combined in a well defined composition.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents. The term “native conformational epitope” or “native proteinepitope” are used interchangeably herein, and include protein epitopesresulting from conformational folding of the integrin molecule whicharise when amino acids from differing portions of the linear sequence ofthe integrin molecule come together in close proximity in 3 dimensionalspace. Such conformational epitopes are distributed on the extracellularside of the plasma membrane.

The term “bispecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has two differentbinding specificities. For example, the molecule may bind to, orinteract with, (a) a cell surface antigen and (b) an Fc receptor on thesurface of an effector cell. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g. aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities. For example, the molecule may bind to,or interact with, (a) a cell surface antigen, (b) an Fc receptor on thesurface of an effector cell, and (c) at least one other component.Accordingly, the invention includes, but is not limited to, bispecific,trispecific, tetraspecific, and other multispecific molecules which aredirected to tissue factor, and to other targets, such as Fc receptors oneffector cells.

The term “bispecific antibodies” also includes diabodies. Diabodies arebivalent, bispecific antibodies in which the VH and VL domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g.,Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;Poljak, R. J., et al. (I 994) Structure 2:1121-1123). Bispecific,heterospecific, heteroconjugate or similar antibodies can also be usedthat are monoclonal, preferably human or humanized, antibodies that havebinding specificities for at least two different antigens. In thepresent case, one of the binding specificities is for at least onetissue factor protein, the other one is for any other antigen. Methodsfor making bispecific antibodies are known in the art. Traditionally,the recombinant production of bispecific antibodies is based on theco-expression of two immunoglobulin heavy chain-light chain pairs, wherethe two heavy chains have different specificities (Milstein and Cuello,Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453,6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985,5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549,4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986),each entirely incorporated herein by reference.

As used herein, the term “heteroantibodies” refers to two or moreantibodies, antibody binding fragments (e.g., Fab), derivativestherefrom, or antigen binding regions linked together, at least two ofwhich have different specificities. These different specificitiesinclude a binding specificity for an Fc receptor on an effector cell,and a binding specificity for an antigen or epitope on a target cell,e.g., a tumor cell.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with a dissociationconstant (K_(D)) of 10⁻⁷ M or less, and binds to the predeterminedantigen with a K_(D) that is at least twofold less than its K_(D) forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”.

As used herein, the term “high affinity” for an IgG antibody refers toan antibody having a K_(D) Of 10⁻⁸ M or less, more preferably 10⁻⁹ M orless and even more preferably 10⁻¹⁰ M or less. However, “high affinity”binding can vary for other antibody isotypes. For example, “highaffinity” binding for an IgM isotype refers to an antibody having aK_(D) of 10⁻⁷ M or less, more preferably 10⁻⁸ M or less. The term“Kassoc” or “Ka”, as used herein, is intended to refer to theassociation rate of a particular antibody-antigen interaction, whereasthe term “Kdis” or “Kd,” as used herein, is intended to refer to thedissociation rate of a particular antibody-antigen interaction, The term“K_(D)”, as used herein, is intended to refer to the dissociationconstant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) andis expressed as a molar concentration (M).

As used herein, “isotype” refers to the antibody class (IgA, IgD, IgE,IgG, or IgM) that is encoded by heavy chain constant region genes. Amonghuman IgG isotypes there are four subclasses; IgG1, IgG2, IgG3 and IgG4named in order of their natural abundance in serum starting from highestto lowest. IgA antibodies are found as two subclasses, IgA1 and IgA2. Asused herein, “isotype switching” also refers to a change between IgGsubclasses or subtypes.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA. The term “isolated nucleic acid molecule,” as used herein inreference to nucleic acids encoding antibodies or antibody portions(e.g., VH, VL, CDR3) that bind to tissue factor, is intended to refer toa nucleic acid molecule in which the nucleotide sequences encoding theantibody or antibody portion are free of other nucleotide sequencesencoding antibodies or antibody portions that bind antigens other thantissue factor, which other sequences may naturally flank the nucleicacid in human genomic DNA. In one embodiment, the anti-tissue factorantibody, or portion thereof, includes the nucleotide or amino acidsequence of CNTO 860.

As disclosed and claimed herein, the invention includes antibodieshaving “conservative sequence modifications” of the sequences set forthin SEQ ID NOs. 2 and 4, i.e., amino acid sequence modifications which donot significantly affect or alter the binding characteristics of theantibody encoded by the nucleotide sequence or containing the amino acidsequence. Such conservative sequence modifications include amino acidsubstitutions, additions and deletions. Codon substitutions of thecoding sequence are often desirable when the expression system for theantibody is altered, e.g. from a murine myeloma cell line to an E. colisystem. Conservative amino acid substitutions include ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a tissue factor antibody ispreferably replaced with another amino acid residue from the same sidechain family.

Modifications can be introduced into the nucleotide sequences of SEQ IDNOs: 1 and 3 by standard techniques known in the art, such assite-directed mutagenesis and PCR-mediated mutagenesis. Codonsubstitutions in SEQ ID NOs: 1 and 3 which do not alter the sequence ofthe encoded protein are also included in the present invention.Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a anti-tissue factor antibody codingsequence, such as by saturation mutagenesis, and the resulting modifiedanti-tissue factor antibodies can be screened for binding activity.

Accordingly, antibodies encoded by the nucleotide sequences disclosedherein and/or containing the amino acid sequences disclosed herein(i.e., SEQ ID NOs: 1-4) include substantially similar antibodies encodedby or containing similar sequences which have been conservativelymodified. Further discussion as to how such substantially similarantibodies can be generated based on the sequences disclosed herein asSEQ ID NOs: 2 and 4 is provided below. The percent identity between twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available atwww.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50,60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percentidentity between two nucleotide or amino acid sequences can alsodetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM 1 20 weight residue table, a gaplength penalty of 12 and a gap penalty of 4. In addition, the percentidentity between two amino acid sequences can be determined using theNeedleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm whichhas been incorporated into the GAP program in the GCG software package(www.gcg.com), using either a Blossum 62 matrix or a PAM2 5 0 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. The nucleic acid and protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.Such searches can be performed using the NBLAST and XBLAST programs(version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215.403-1 0.BLAST nucleotide searches can be performed with the NBLAST program,score=100, wordlength=12 to obtain nucleotide sequences homologous tothe nucleic acid molecules of the invention. BLAST protein searches canbe performed with the XBLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to the protein molecules of theinvention. To obtain gapped alignments for comparison purposes, GappedBLAST can be utilized as described in Altschul et al., (1997) NucleicAcids Res. 25(17):3389 When utilizing BLAST and Gapped BLAST programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof, may be mutated inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, CHO cells andlymphocytic cells.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and nonmammals, such as nonhuman primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

CITATIONS

All publications or patents cited herein are entirely incorporatedherein by reference as they show the state of the art at the time of thepresent invention and/or to provide description and enablement of thepresent invention. Publications refer to any scientific or patentpublications, or any other information available in any media format,including all recorded, electronic or printed formats. The followingreferences are entirely incorporated herein by reference: Ausubel, etal., ed., Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, N.Y. (1987-2001); Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y. (1989);Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor,N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology,John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., CurrentProtocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

1. Production of Antibodies

Anti-tissue factor antibodies of the present invention can be optionallyproduced by a variety of techniques, including conventional monoclonalantibody techniques, e.g., the standard somatic cell hybridizationtechnique of Kohler and Milstein (1975) Nature 256:495. A variety ofcell lines, mixed cell lines, an immortalized cell or clonal populationof immortalized cells, can be used, as well known in the art.

Antibodies that are specific for human tissue factor proteins orfragments thereof can be raised against an appropriate immunogenicantigen, such as isolated and/or tissue factor protein or a portionthereof (including synthetic molecules, such as synthetic peptides).Other specific or general mammalian antibodies can be similarly raised.Preparation of immunogenic antigens, and monoclonal antibody productioncan be performed using any suitable technique.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0,Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1, JURKAT, WEHI,K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO,PerC.6, YB2/O or the like, or heteromyelomas, fusion products thereof,or any cell or fusion cell derived therefrom, or any other suitable cellline as known in the art. See, e.g., www.atcc.org, www.lifetech.com.,and the like, with antibody producing cells, such as, but not limitedto, isolated or cloned spleen, peripheral blood, lymph, tonsil, or otherimmune or B cell containing cells, or any other cells expressing heavyor light chain constant or variable or framework or CDR sequences,either as endogenous or heterologous nucleic acid, as recombinant orendogenous, viral, bacterial, algal, prokaryotic, amphibian, insect,reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference. Antibody producing cells canalso be obtained from the peripheral blood or, preferably the spleen orlymph nodes, of humans or other suitable animals that have beenimmunized with the antigen of interest. Any other suitable host cell canalso be used for expressing heterologous or endogenous nucleic acidencoding an antibody, specified fragment or variant thereof, of thepresent invention. The fused cells (hybridomas) or recombinant cells canbe isolated using selective culture conditions or other suitable knownmethods, and cloned by limiting dilution or cell sorting, or other knownmethods. Cells which produce antibodies with the desired specificity canbe selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, Del.; Biovation, Aberdeen, Scotland, UK;Bioinvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., EP 368,684, PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260(May 12, 1994); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; (Scripps); EP 614 989(MorphoSys); WO95/16027 (Bioinvent); WO88/06630; WO90/3809 (Dyax); U.S.Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323,5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803, EP590 689 (Ixsys, now Applied Molecular Evolution (AME), each entirelyincorporated herein by reference) or that rely upon immunization oftransgenic animals (e.g., SCID mice, Nguyen et al., Microbiol. Immunol.41:901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118(1996); Eren et al., Immunol. 93:154-161 (1998), each entirelyincorporated by reference as well as related patents and applications)that are capable of producing a repertoire of human antibodies, as knownin the art and/or as described herein. Such techniques, include, but arenot limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci.USA, 94:4937-4942 (May 1997); Hanes et al., Proc. Natl. Acad. Sci. USA,95:14130-14135 (November 1998)); single cell antibody producingtechnologies (e.g., selected lymphocyte antibody method (“SLAM”) (U.S.Pat. No. 5,627,052, Wen et al., J. Immunol. 17:887-892 (1987); Babcooket al., Proc. Natl. Acad. Sci. USA 93:7843-7848 (1996)); gelmicrodroplet and flow cytometry (Powell et al., Biotechnol. 8:333-337(1990); One Cell Systems, Cambridge, Mass.; Gray et al., J. Imm. Meth.182:155-163 (1995); Kenny et al., Bio/Technol. 13:787-790 (1995));B-cell selection (Steenbakkers et al., Molec. Biol. Reports 19:125-134(1994); Jonak et al., Progress Biotech, Vol. 5, In Vitro Immunization inHybridoma Technology, Borrebaeck, ed., Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies canalso be used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcewhich is non-human, e.g., but not limited to mouse, rat, rabbit,non-human primate or other mammal. These human amino acid residues areoften referred to as “import” residues, which are typically taken froman “import” variable, constant or other domain of a known humansequence. Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast;www.atcc.org/phage/hdb.html; www.kabatdatabase.com/top.html;www.antibodyresource.com/onlinecomp.html; www.appliedbiosystems.com;www.biodesign.com; antibody.bath.ac.uk; http://www.unizh.ch/˜antibody/;www.cryst.bbk.ac.uk/˜ubcg07s; Kabat et al., Sequences of Proteins ofImmunological Interest, U.S. Dept. Health (1983), each entirelyincorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. Generally part or all of the non-human or human CDRsequences are maintained while the non-human sequences of the variableand constant regions are replaced with human or other amino acids.Antibodies can also optionally be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR (framework) residues can be selected and combined from theconsensus and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. Humanization orengineering of antibodies of the present invention can be performedusing any known method, such as but not limited to those described in,Winter (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)), Sims et al.,J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901(1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992);Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat. Nos. 5,723,323,5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323,5,766886, 5714352, 6204023, 6180370, 5693762, 5530101, 5585089, 5225539;4816567, PCT: US98/16280, US96/18978, US91/09630, US91/05939,US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424,WO90/14430, EP 229246, each entirely incorporated herein by reference,included references cited therein.

The tissue factor antibody can also be optionally generated byimmunization of a transgenic animal (e.g., mouse, rat, hamster,non-human primate, and the like) capable of producing a repertoire ofhuman antibodies, as described herein and/or as known in the art. Cellsthat produce a human tissue factor antibody can be isolated from suchanimals and immortalized using suitable methods, such as the methodsdescribed herein.

Transgenic mice that can produce a repertoire of human antibodies thatbind to human antigens can be produced by known methods (e.g., but notlimited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126,5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al.;Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg etal. WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585,Kucherlapate et al. WO 96/34096, Kucherlapate et al. EP 0463 151 B1,Kucherlapate et al. EP 0710 719 A1, Surani et al. U.S. Pat. No.5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438 474B1, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440 A,Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int. Immunol.6(4)579-591 (1994), Green et al, Nature Genetics 7:13-21 (1994), Mendezet al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic AcidsResearch 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad SciUSA 90(8)3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1):65-93(1995) and Fishwald et al., Nat Biotechnol 14(7):845-851 (1996), whichare each entirely incorporated herein by reference). Generally, thesemice comprise at least one transgene comprising DNA from at least onehuman immunoglobulin locus that is functionally rearranged, or which canundergo functional rearrangement. The endogenous immunoglobulin loci insuch mice can be disrupted or deleted to eliminate the capacity of theanimal to produce antibodies encoded by endogenous genes.

To generate hybridomas producing human monoclonal antibodies to tissuefactor, splenocytes and lymph node cells from immunized mice can beisolated and fused to an appropriate immortalized cell line, such as amouse myeloma cell line. The resulting hybridomas can be screened forthe production of antigen-specific antibodies.

Human antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(e.g., Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification, site directed mutagenesis) and can be inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the VH segmentis operatively linked to the CH segment(s) within the vector and the VI,segment is operatively linked to the CL segment within the vector.Additionally or alternatively, the recombinant expression vector canencode a signal peptide that facilitates secretion of the antibody chainfrom a host cell. The antibody chain gene can be cloned into the vectorsuch that the signal peptide is linked in-frame to the amino terminus ofthe antibody chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody. Prokaryoticexpression of antibody genes has been reported to be ineffective forproduction of high yields of active antibody (Boss, M. A. and Wood, C.R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfrCHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:42164220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (I 982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. In particular,for use with NSO myeloma cells, another preferred expression system isthe GS gene expression system disclosed in WO 87/04462, WO 89/01036 andEP 338,841. When recombinant expression vectors encoding antibody genesare introduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or, more preferably,secretion of the antibody into the culture medium in which the hostcells are grown. Antibodies can be recovered from the culture mediumusing standard protein purification methods.

Screening antibodies for specific binding to similar proteins orfragments can also be conveniently achieved using peptide displaylibraries. This method involves the screening of large collections ofpeptides for individual members having the desired function orstructure. Antibody screening of peptide display libraries is well knownin the art. The displayed peptide sequences can be from 3 to 5000 ormore amino acids in length, frequently from 5-100 amino acids long, andoften from about 8 to 25 amino acids long. In addition to directchemical synthetic methods for generating peptide libraries, severalrecombinant DNA methods have been described. One type involves thedisplay of a peptide sequence on the surface of a bacteriophage or cell.Each bacteriophage or cell contains the nucleotide sequence encoding theparticular displayed peptide sequence. Such methods are described in PCTPatent Publication Nos. 91/17271, 91/18980, 91/19818, and 93/08278.Other systems for generating libraries of peptides have aspects of bothin vitro chemical synthesis and recombinant methods. See, PCT PatentPublication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Pat.Nos. 5,658,754; and 5,643,768. Peptide display libraries, vector, andscreening kits are commercially available from such suppliers asInvitrogen (Carlsbad, Calif.), and Cambridge antibody Technologies(Cambridgeshire, UK). See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666,4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730,5,763,733, 5,767,260, 5,856,456, assigned to Enzon; 5,223,409,5,403,484, 5,571,698, 5,837,500, assigned to Dyax, 5,427,908, 5,580,717,assigned to Affymax; 5,885,793, assigned to Cambridge antibodyTechnologies; 5,750,373, assigned to Genentech, 5,618,920, 5,595,898,5,576,195, 5,698,435, 5,693,493, 5,698,417, assigned to Xoma, Colligan,supra; Ausubel, supra; or Sambrook, supra, each of the above patents andpublications entirely incorporated herein by reference.

Antibodies of the present invention can also be prepared using at leastone tissue factor antibody encoding nucleic acid to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, and the like,that produce such antibodies in their milk. Such animals can be providedusing known methods. See, e.g., but not limited to, U.S. Pat. Nos.5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362;5,304,489, and the like, each of which is entirely incorporated hereinby reference.

Antibodies of the present invention can additionally be prepared usingat least one tissue factor antibody encoding nucleic acid to providetransgenic plants and cultured plant cells (e.g., but not limited totobacco, maize, and duckweed) that produce such antibodies, specifiedportions or variants in the plant parts or in cells cultured therefrom.As a non-limiting example, transgenic tobacco leaves expressingrecombinant proteins have been successfully used to provide largeamounts of recombinant proteins, e.g., using an inducible promoter. See,e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencecited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to know methods. See also,e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October,1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., PlantPhysiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans.22:940-944 (1994); and references cited therein. See, also generally forplant expression of antibodies, but not limited to, Each of the abovereferences is entirely incorporated herein by reference.

2. Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencesencoding at least 70-100% of the contiguous amino acids of at least oneof SEQ ID NOS: 5 and 6, specified fragments, variants or consensussequences thereof, or a deposited vector comprising at least one ofthese sequences, a nucleic acid molecule of the present inventionencoding at least one anti-tissue factor antibody can be obtained usingmethods described herein or as known in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising the coding sequence for, but notlimited to, at least one specified portion of at least one CDR, as CDR1,CDR2 and/or CDR3 of at least one heavy chain (e.g., SEQ ID NOS: 7-9) orlight chain (e.g., SEQ ID NOS: 10-12); nucleic acid molecules comprisingthe coding sequence for an anti-tissue factor antibody or variableregion (e.g., SEQ ID NOS: 2, 4, 5 and 6) including but not limited toSEQ ID Nos; 1 and 3; and nucleic acid molecules which comprise anucleotide sequence substantially different from those described abovebut which, due to the degeneracy of the genetic code, still encode atleast one anti-tissue factor antibody as described herein and/or asknown in the art. Of course, the genetic code is well known in the art.Thus, it would be routine for one skilled in the art to generate suchdegenerate nucleic acid variants that code for specific anti-tissuefactor antibodies of the present invention. See, e.g., Ausubel, et al.,supra, and such nucleic acid variants are included in the presentinvention. Non-limiting examples of isolated nucleic acid molecules ofthe present invention include SEQ ID NOS: 5, 13-15, and 6, 16-18corresponding to non-limiting examples of a nucleic acid encoding,respectively, HC variable region, HC CDR1, HC CDR2, HC CDR3, and LCvariable region LC CDR1, LC CDR2, LC CDR3.

In another aspect, the invention provides isolated nucleic acidmolecules encoding a(n) anti-tissue factor subunit antibody having anamino acid sequence as encoded by the nucleic acid contained in theplasmid designated clone p2401 and p2402.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-tissue factor antibodycan include, but are not limited to, those encoding the amino acidsequence of an antibody fragment, by itself, the coding sequence for theentire antibody or a portion thereof, the coding sequence for anantibody, fragment or portion, as well as additional sequences, such asthe coding sequence of at least one signal leader or fusion peptide,with or without the aforementioned additional coding sequences, such asat least one intron, together with additional, non-coding sequences,including but not limited to, non-coding 5′ and 3′ sequences, such asthe transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals (for example—ribosome binding and stability of mRNA); anadditional coding sequence that codes for additional amino acids, suchas those that provide additional functionalities. Thus, the sequenceencoding an antibody can be fused to a marker sequence, such as asequence encoding a peptide that facilitates purification of the fusedantibody comprising an antibody fragment or portion.

3. Polynucleotides which Selectively Hybridize to a Polynucleotide asDescribed Herein

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides of the present inventioncan be used to identify, isolate, or amplify partial or full-lengthclones in a deposited library. In some embodiments, the polynucleotidesare genomic or cDNA sequences isolated, or otherwise complementary to, acDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably at least 85% or 90% full-length sequences, andmore preferably at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of an antibody encoded by the polynucleotides described herein.The polynucleotides of this invention embrace nucleic acid sequencesthat can be employed for selective hybridization to a polynucleotideencoding an antibody of the present invention. See, e.g., Ausubel,supra; Colligan, supra, each entirely incorporated herein by reference.

4. Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra)

5. Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

6. Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; 4,795,699 and 4,921,794 to Tabor, et al; 5,142,033 toInnis; 5,122,464 to Wilson, et al.; 5,091,310 to Innis; 5,066,584 toGyllensten, et al; 4,889,818 to Gelfand, et al; 4,994,370 to Silver, etal; 4,766,067 to Biswas; 4,656,134 to Ringold) and RNA mediatedamplification that uses anti-sense RNA to the target sequence as atemplate for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 toMalek, et al, with the tradename NASBA), the entire contents of whichreferences are incorporated herein by reference. (See, e.g., Ausubel,supra; or Sambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can beused to improve yield of long PCR products.

7. Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences. Such a method ofconstructing functional dsDNA molecules is taught in U.S. Pat. No.6,521,427 and WO02081490.

8. Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example a cDNA or a genomicsequence encoding an antibody of the present invention, can be used toconstruct a recombinant expression cassette that can be introduced intoat least one desired host cell. A recombinant expression cassette willtypically comprise a polynucleotide of the present invention operablylinked to transcriptional initiation regulatory sequences that willdirect the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in intron) of a non-heterologous formof a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to includes promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or P-globin promoter.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

9. Vectors and Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one anti-tissue factor antibody by recombinant techniques,as is well known in the art. See, e.g., Sambrook, et al., supra;Ausubel, et al., supra, each entirely incorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one antibody of the present invention can be expressed in amodified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of an antibody to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage. Also, peptide moieties can beadded to an antibody of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofan antibody or at least one fragment thereof. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Chapters17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention.

Alternatively, nucleic acids of the present invention can be expressedin a host cell by turning on (by manipulation) in a host cell thatcontains endogenous DNA encoding an antibody of the present invention.Such methods are well known in the art, e.g., as described in U.S. Pat.Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirelyincorporated herein by reference.

Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, PerC.6 cells, hep G2 cells,P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells and the like, which arereadily available from, for example, American Type Culture Collection,Manassas, Va. (www.atcc.org). Preferred host cells include cells oflymphoid origin such as myeloma and lymphoma cells.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art. Also, to avoid high surface expression of heavy chainmolecules, it may be necessary to use an expression vector thateliminates transmembrane domain variant splices.

10. Purification of an Antibody

A tissue factor antibody can be recovered and purified from recombinantcell cultures by well-known methods including, but not limited to,protein A purification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

11. Anti-Tissue Factor Antibodies of the Invention

Since it is well known in the art that antibody heavy and light chainCDR3 domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen, the recombinantantibodies of the invention prepared as set forth above preferablycomprise the heavy and light chain CDR3s of CNTO 860. The antibodiesfurther can comprise the CDR2s of CNTO 860. The antibodies further cancomprise the CDR1s of CNTO 860. Accordingly, the invention furtherprovides anti-tissue factor antibodies comprising: (1) human heavy chainframework regions, a human heavy chain CDR1 region, a human heavy chainCDR2 region, and a human heavy chain CDR3 region, wherein the humanheavy chain CDR3 region is as shown in SEQ ID NO: 9, and (2) human lightchain framework regions, a human light chain CDR1 region, a human lightchain CDR2 region, and a human light chain CDR3 region, wherein thehuman light chain CDR3 region is as shown in SEQ ID NO: 12, and (3) anFc receptor binding domain; wherein the antibody binds human tissuefactor and an Fc receptor on the surface of an effector cell. Theantibody may further comprise the heavy chain CDR2 and/or the lightchain CDR2 of CNTO 860, as shown in SEQ ID NO: 8 and/or 11,respectively. The antibody may further comprise the heavy chain CDR1and/or the light chain CDR1 of CNTO 860, as shown in SEQ ID NO: 7 and/or10, respectively.

In a preferred embodiment the three heavy chain CDRs and the three lightchain CDRs of the antibody or antigen-binding fragment have the aminoacid sequence of the corresponding CDR of CNTO 860, as described herein.Such antibodies can be prepared by chemically joining together thevarious portions (e.g., CDRs and framework portions, FR1, FR2, FR3, andFR4) of the antibody using conventional techniques, by preparing andexpressing a (i.e., one or more) nucleic acid molecule that encodes theantibody using conventional techniques of recombinant DNA technology orby using any other suitable method.

Preferably, the CDR1, 2, and/or 3 of the engineered antibodies describedabove comprise the exact amino acid sequence(s) as those of CNTO 860disclosed herein. However, the ordinarily skilled artisan willappreciate that some deviation from the exact CDR sequences of CNTO 860may be possible while still retaining the ability of the antibody tobind human tissue factor effectively (e.g., conservative substitutions).Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDRs that are, for example, 90%, 95%, 98% or99.5% identical to one or more CDRs of CNTO 860. In addition to bindingtissue factor as well as an Fc receptor. Engineered antibodies such asthose described above may be selected for their retention of otherfunctional properties of antibodies of the invention, such as:

1). binding to live cells expressing human tissue factor; 2) binding tohuman tissue factor with a K_(D) of 10⁻⁸ M or less (e.g., 10⁻⁹ M or10⁻¹⁰ M or less); 3) binding to the unique epitope on tissue factorrecognized by the TF8-5G9 antibody (to eliminate the possibility thatmonoclonal antibodies with complimentary activities when used incombination would compete for binding to the same epitope); 4) bindingto an Fc receptor and is capable of eliciting effector functionsincluding ADCC, complement activation, and C1q binding; and 4)inhibition of the growth of tumor cells in vivo.

Human monoclonal antibodies of the invention can be tested for bindingto tissue factor by, for example, standard ELISA.

To determine if the selected human anti-tissue factor monoclonalantibodies bind to unique epitopes, each antibody can be biotinylatedusing commercially available reagents (Pierce, Rockford, Ill.).Competition studies using unlabeled monoclonal antibodies andbiotinylated monoclonal antibodies can be performed using tissue factorcoated-ELISA plates. Biotinylated mAb binding can be detected with astrep-avidin-alkaline phosphatase probe. To determine the isotype ofpurified antibodies, isotype ELISAs can be performed. In order todemonstrate binding of monoclonal antibodies to live cells expressingthe tissue factor, flow cytometry can be used. Anti-tissue factor humanIgGs can be further tested for reactivity with tissue factor antigen byWestern blotting.

An antibody of the invention can be of any class (IgG, IgA, IgM, etc.)that contains an Fc receptor binding domain and thus has the desiredspectrum of effector functions conferred by that isotype and subclassand can comprise a kappa or lambda light chain. Quantifiable propertiesof antibody isotypes and subclasses thought to confer in vivo activitiessuch as ADCC, CA, and opsinization are shown below and described in e.g.Janeway et al. eds., 2001. Immunobiology 5: The immune system in healthan disease, Garland Publishing, NY, N.Y., USA. Chapters 4 and 9. In oneembodiment, the antibody comprises an IgG heavy chain or definedfragment, for example, at least one of isotypes, IgG1, IgG2, or IgG3,preferably an IgG1 class. In another embodiment, the anti-human tissuefactor antibody comprises an IgG1 heavy chain and a IgG1 light chain.

IgG1 IgG2 IgG3 IgG4 IgM IgA IgE IgD Complement Activation ++ + +++ −+++ + − − Phagocyte Binding + − + +/− − − − − Neutralization ++ ++ ++ ++++ + − − Opsinization +++ +/− ++ + − + − − Sensitization for killing byNKs ++ − ++ − − − − − Sensitization of Mast cells + − + − − − − +++Extravascular diffusion +++ +++ +++ +++ +/− ++ − + (sIgA)

In another aspect of the invention, the structural features of an humananti-tissue factor antibodies of the invention, CNTO 860, are used tocreate structurally related human anti-tissue factor antibodies thatretain the functional properties of the antibodies of the invention,i.e. the binding to human tissue factor and an Fc receptor.

The antibodies of the invention can bind human tissue factor with a widerange of affinities (K_(D)). In a preferred embodiment, at least onehuman mAb of the present invention can optionally bind human tissuefactor with high affinity. For example, a human mAb can bind humantissue factor with a K_(D) equal to or less than about 10⁻⁷ M, such asbut not limited to, 0.1-9.9 (or any range or value therein)×10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ M or any range or value therein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

Preferably, the antibody or antigen-binding fragment of the inventionbinds human tissue factor and, thereby partially or substantiallyneutralizes at least one biological activity of the protein. Anantibody, or specified portion or variant thereof, that partially orpreferably substantially neutralizes at least one biological activity ofat least one tissue factor protein or fragment can bind the protein orfragment and thereby inhibit activities mediated through the binding oftissue factor to its ligand or through other tissue factor-dependent ormediated mechanisms. As used herein, the term “neutralizing antibody”refers to an antibody that can inhibit a tissue factor-dependentactivity by about 20-120%, preferably by at least about 10, 20, 30, 40,50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100% or more depending on the assay. The capacity of an anti-tissuefactor antibody to inhibit a tissue factor-dependent activity ispreferably assessed by at least one suitable tissue factor protein orreceptor assay, as described herein and/or as known in the art.

In a specific embodiment, the anti-tissue factor antibody comprises anantibody having the amino acid sequence of the light chain of SEQ ID No.4 and the amino acid sequence of the heavy chain of SEQ ID NO. 2.

An anti-tissue factor antibody of the present invention can include oneor more amino acid substitutions, deletions or additions, either fromnatural mutations or human manipulation, as specified herein. Thus, theinvention also relates to antibodies, antigen-binding fragments,immunoglobulin chains and CDRs comprising amino acids in a sequence thatis substantially the same as an amino acid sequence described herein.Preferably, such antibodies or antigen-binding fragments and antibodiescomprising such chains or CDRs can bind human tissue factor with highaffinity (e.g., K_(D) less than or equal to about 10⁻⁹ M). Amino acidsequences that are substantially the same as the sequences describedherein include sequences comprising conservative amino acidsubstitutions, as well as amino acid deletions and/or insertions. Aconservative amino acid substitution refers to the replacement of afirst amino acid by a second amino acid that has chemical and/orphysical properties (e.g, charge, structure, polarity,hydrophobicity/hydrophilicity) that are similar to those of the firstamino acid. Conservative substitutions include replacement of one aminoacid by another within the following groups: lysine (K), arginine (R)and histidine (H); aspartate (D) and glutamate (E); asparagine (N),glutamine (Q), serine (S), threonine (T), tyrosine (Y), K, R, H, D andE; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P),phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) andglycine (G); F, W and Y; C, S and T.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for any given anti-tissue factor antibody will not be morethan 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, 1, such as 1-30 or any range or value therein, as specifiedherein.

Amino acids in an anti-tissue factor antibody of the present inventionthat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells,Science 244:1081-1085 (1989)). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to at least one tissue factor neutralizing activity. Sites thatare critical for antibody binding can also be identified by structuralanalysis such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992)and de Vos, et al., Science 255:306-312 (1992)).

Anti-tissue factor antibodies of the present invention can include, butare not limited to, at least one portion, sequence or combinationselected from 5 to all of the contiguous amino acids of at least one ofSEQ ID NOS: 2 and 4.

A(n) anti-tissue factor subunit antibody can further optionally comprisea polypeptide of at least one of 70-100% of the contiguous amino acidsof at least one of SEQ ID NOS: 5 and 6 and an Fc binding portion.

In one embodiment, the amino acid sequence of an immunoglobulin chain,or portion thereof (e.g., variable region, CDR) has about 70-100%identity (e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 orany range or value therein) to the amino acid sequence of thecorresponding chain of at least one of SEQ ID NOS: 2 and 4. For example,the amino acid sequence of a light chain variable region can be comparedwith the sequence of SEQ ID NO: 6, or the amino acid sequence of a heavychain CDR3 can be compared with SEQ ID NO: 9. Preferably, 70-100% aminoacid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or anyrange or value therein) is determined using a suitable computeralgorithm, as known in the art.

Exemplary heavy chain and light chain variable regions sequences areprovided in SEQ ID NOS: 5 and 6. The antibodies of the presentinvention, or specified variants thereof, can comprise any number ofcontiguous amino acid residues from an antibody of the presentinvention, wherein that number is selected from the group of integersconsisting of from 10-100% of the number of contiguous residues in ananti-tissue factor antibody. Optionally, this subsequence of contiguousamino acids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,110, 120 or more amino acids in length, or any range or value therein.Further, the number of such subsequences can be any integer selectedfrom the group consisting of from 1 to 20, such as at least 2, 3, 4, or5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and preferably at least 50%, 60%, or 70%, and mostpreferably at least 80%, 90%, or 95%-100% of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity, are well known to those of skill in the art.

In another aspect, the invention relates to antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

The modified antibodies and antigen-binding fragments of the inventioncan comprise one or more organic moieties that are covalently bonded,directly or indirectly, to the antibody. Each organic moiety that isbonded to an antibody or antigen-binding fragment of the invention canindependently be a hydrophilic polymeric group, a fatty acid group or afatty acid ester group. As used herein, the term “fatty acid”encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, anantibody modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying antibodies of the invention can be linear or branched andinclude, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodies ofthe invention include, for example, n-dodecanoate (C₁₂, laurate),n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈, stearate),n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂, behenate),n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀), cis-Δ9-octadecanoate(C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate (C₂₀, arachidonate),octanedioic acid, tetradecanedioic acid, octadecanedioic acid,docosanedioic acid, and the like. Suitable fatty acid esters includemono-esters of dicarboxylic acids that comprise a linear or branchedlower alkyl group. The lower alkyl group can comprise from one to abouttwelve, preferably one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis(Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996);Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and themethods described in Hermanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

12. Anti-Tissue Factor Antibody Compositions

The present invention also provides at least one anti-tissue factorantibody composition comprising at least one tissue factor antibody asdescribed herein and/or as known in the art that are provided in anon-naturally occurring composition, mixture or form. Anti-tissue factorantibody compounds, compositions or combinations of the presentinvention can further comprise at least one of any suitable auxiliary,such as, but not limited to, diluent, binder, stabilizer, buffers,salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the anti-tissue factor antibody, fragment or variantcomposition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/antibody components, which canalso function in a buffering capacity, include alanine, glycine,arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine,lysine, leucine, isoleucine, valine, methionine, phenylalanine,aspartame, and the like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Anti-tissue factor antibody compositions can also include a buffer or apH adjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid saltssuch as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts such as citrate.

Additionally, anti-tissue factor subunit antibody compositions of theinvention can include polymeric excipients/additives such aspolyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g.,cyclodextrins, such as 2-hydroxypropyl-α-cyclodextrin), polyethyleneglycols, flavoring agents, antimicrobial agents, sweeteners,antioxidants, antistatic agents, surfactants (e.g., polysorbates such as“TWEEN 20” and “TWEEN 80”), lipids (e.g., phospholipids, fatty acids),steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the anti tissue factor antibody, portion or variantcompositions according to the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed.,Williams & Williams, (1995), and in the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), the disclosuresof which are entirely incorporated herein by reference. Preferredcarrier or excipient materials are carbohydrates (e.g., saccharides andalditols) and buffers (e.g., citrate) or polymeric agents.

Anti-tissue factor antibody compositions of the present invention canoptionally further comprise at least one additional agent selected fromat least one TNF antagonist (e.g., but not limited to a TNF antibody orfragment, a soluble TNF receptor or fragment, fusion proteins thereof,or a small molecule TNF antagonist), an antirheumatic (e.g.,methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, goldsodium thiomalate, hydroxychloroquine sulfate, leflunomide,sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anethetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a fluororquinolone, a macrolide, apenicillin, a sulfonamide, a tetracycline, another antimicrobial), anantipsoriatic, a corticosteroid, (dexamethasone), an anabolic steroid(testosterone), a diabetes related agent, a mineral, a nutritional, athyroid agent, a vitamin, a calcium related hormone, an antidiarrheal,an antitussive, an antiemetic, an antiulcer, a laxative, ananticoagulant, an erythropoietin (e.g., epoetin alpha), a filgrastim(e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin (rituximab), an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormoneantagonist, a reproductive hormone antagonist (flutamide, nilutamide), ahormone release modulator (leuprolide, goserelin), a hormone replacementdrug, an estrogen receptor modulator (tamoxifen), a retinoid(tretinoin), a topoisomerase inhibitor (etoposide, irinotecan), acytoxin (doxorubicin, dacarbazine), a mydriatic, a cycloplegic, analkylating agent (carboplatin), a nitrogen mustard (melphalen,chlorabucil), a nitrosourea (carmustine, estramustine) an antimetabolite(methotrexate, cytarabine, fluorouracil), a mitotic inhibitor(vincristine, taxol), a radiopharmaceutical (Iodine 131-tositumomab), aradiosensitizer (misonidazole, tirapazamine) an antidepressant,antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, asympathomimetic, a stimulant, donepezil, tacrine, an asthma medication,a beta agonist, an inhaled steroid, a leukotriene inhibitor, amethylxanthine, a cromolyn, an epinephrine or analog, dornase alpha(Pulmozyme), a cytokine (interferon alpha-2, IL2) or a cytokineantagonist (infliximab). Non-limiting examples of such cytokinesinclude, but are not limited to, any of IL-1 to IL-23, IL-6, anti-tumorantibodies, chemotherapeutic agents or radiation therapies. Suitabledosages are well known in the art. See, e.g., Wells et al., eds.,Pharmacotherapy Handbook, 2^(nd) Edition, Appleton and Lange, Stamford,Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000,Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), each ofwhich references are entirely incorporated herein by reference.

The method may be carried out by combining the TF antagonists of theinvention with one or more other agents having anti-tumor effect or adissimilar mechanism of inhibiting in vivo tumor growth, including, butnot limited to chemotherapeutic agents.

Further, the TF antibody can be combined with one or moreanti-angiogenic agents. Angiogenesis is characterized by the invasion,migration and proliferation of smooth muscle and endothelial cells. Theαvβ3 integrin (also known as the vitronectin receptor) is known to playa role in various conditions or disease states including tumormetastasis, solid tumor growth (neoplasia), osteoporosis, Paget'sdisease, humoral hypercalcemia of malignancy, angiogenesis, includingtumor angiogenesis, retinopathy, including macular degeneration,arthritis, including rheumatoid arthritis, periodontal disease,psoriasis and smooth muscle cell migration (e.g. restenosis).

The adhesion receptor integrin αvβ3 binds vitronectin, fibrinogen, vonWillebrand Factor, laminin, thrombospondin, and other like ligands. Itwas identified as a marker of angiogenic blood vessels in chick and manand plays a critical role in angiogenesis or neovascularization.Antagonists of αvβ3 inhibit this process by selectively promotingapoptosis of cells in neovasculature. Therefore, αvβ3 antagonists wouldbe useful therapeutic targets for treating such conditions associatedwith neovascularization (Brooks et al., Science, Vol. 264, (1994),569-571). Additionally, tumor cell invasion occurs by a three stepprocess: 1) tumor cell attachment to extracellular matrix; 2)proteolytic dissolution of the matrix; and 3) movement of the cellsthrough the dissolved barrier. This process can occur repeatedly and canresult in metastases at sites distant from the original tumor. The αvβ3integrin has been shown to play a role in tumor cell invasion as well asangiogenesis.

As the antagonists of αvβ3 and neutralizing anti-TF antibodies bothtarget tumors but act through different mechanisms, the combination ofanti-integrin antibodies with anti-TF antibodies should result in aparticularly potent and effective combination therapy with little normaltissue toxicity. Thus, in one embodiment of the present invention, thereis provided a method of inhibiting the growth of tumors which comprisesadministering a combination of an integrin antagonist and an anti-TFantibody in a patient in need of such treatment. Other antibodies whichselectively bind integrins or integrin subunits, especially those thatbind the alphaV subunit, are disclosed in U.S. Pat. Nos. 5,985,278 and6,160,099. Mabs that inhibit binding of alphaVbeta3 to its naturalligands containing the tripeptide argininyl-glycyl-aspartate (RGD) aredisclosed in U.S. Pat. No. 5,766,591 and WO0078815. Other antibodiesthat prevent alphaV-subunit containing integrins from binding tovitronection, fibronectin, or other ligands have similar utility inpreventing angiogenesis. Such antibodies include the antibody known atGEN 095 or CNTO 95 and described in applicants co-pending applicationpublished as WO02012501.

In accordance with the invention, other known anti-angiogenesis agentssuch as thalidomide may also be employed in combination with ananti-tissue factor antibody.

The invention also pertains to immunoconjugates comprising the antibodydescribed herein conjugated to a cytotoxic agent such as achemotherapeutic agent, toxin (e.g., an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), or aradioactive isotope (i.e., a radioconjugate).

Such anti-cancer can also include toxin molecules that are associated,bound, co-formulated or co-administered with at least one antibody ofthe present invention. The toxin can optionally act to selectively killthe pathologic cell or tissue. The pathologic cell can be a cancer orother cell. Such toxins can be, but are not limited to, purified orrecombinant toxin or toxin fragment comprising at least one functionalcytotoxic domain of toxin, e.g., selected from at least one of ricin,diphtheria toxin, a venom toxin, or a bacterial toxin. The term toxinalso includes both endotoxins and exotoxins produced by any naturallyoccurring, mutant or recombinant bacteria or viruses which may cause anypathological condition in humans and other mammals, including toxinshock, which can result in death. Such toxins may include, but are notlimited to, enterotoxigenic E. coli heat-labile enterotoxin (LT),heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonasenterotoxins, toxic shock syndrome toxin-1 (TSST-1), Staphylococcalenterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins andthe like. Such bacteria include, but are not limited to, strains of aspecies of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli(e.g., strains of serotype 0157:H7), Staphylococcus species (e.g.,Staphylococcus aureus, Staphylococcus pyogenes), Shigella species (e.g.,Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigellasonnei), Salmonella species (e.g., Salmonella typhi, Salmonellacholera-suis, Salmonella enteritidis), Clostridium species (e.g.,Clostridium perfringens, Clostridium dificile, Clostridium botulinum),Camphlobacter species (e.g., Camphlobacter jejuni, Camphlobacter fetus),Heliobacter species, (e.g., Heliobacter pylori), Aeromonas species(e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae),Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species (e.g.,Vibrios cholerae, Vibrios parahemolyticus), Klebsiella species,Pseudomonas aeruginosa, and Streptococci. See, e.g., Stein, ed.,INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston,(1990); Evans et al., eds., Bacterial Infections of Humans: Epidemiologyand Control, 2d. Ed., pp 239-254, Plenum Medical Book Co., New York(1991); Mandell et al Principles and Practice of Infectious Diseases,3d. Ed., Churchill Livingstone, New York (1990); Berkow et al, eds., TheMerck Manual, 16th edition, Merck and Co., Rahway, N.J., 1992; Wood etal, FEMS Microbiology Immunology, 76:121-134 (1991); Marrack et al,Science, 248:705-711 (1990), the contents of which references areincorporated entirely herein by reference.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above.

A variety of radionuclides are available for the production ofradioconjugated anti-TF antibodies.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such as N-succinimidyl(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctionalderivatives of imidoesters (such as dimethyl adipimidate HCL), activeesters (such as disuccinimidyl suberate), aldehydes (such asglutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon labeled 1-isothiocyanatobenzyl methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

The anti-Tissue factor antibodies disclosed herein may also beformulated as immunoliposomes. Liposomes containing the antibody areprepared by methods known in the art, such as described in Epstein etal., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.Natl. Acad. Sci. USA 77:4030 (1980 Particularly useful liposomes can begenerated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter. Fab′ fragments of the antibody of the presentinvention can be conjugated to the liposomes as described in Mar-tin etal., J. Biol. Chem. 257:286-288 (1982) via a disulfide interchangereaction. A chemotherapeutic agent (such as Doxorubicin) is optionallycontained within the liposome. See Gabizon et al., J National CancerInst. 81(19):1484 (1989).

14. Formulations

As noted above, the invention provides for stable formulations, which ispreferably a phosphate buffer with saline or a chosen salt, as well aspreserved solutions and formulations containing a preservative as wellas multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one anti-tissue factor antibody in apharmaceutically acceptable formulation. Preserved formulations containat least one known preservative or optionally selected from the groupconsisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concentration ormixture can be used as known in the art, such as 0.001-5%, or any rangeor value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1-2%m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol(e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g.,0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9,1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5,0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one anti-tissue factor subunit antibody with theprescribed buffers and/or preservatives, optionally in an aqueousdiluent, wherein said packaging material comprises a label thatindicates that such solution can be held over a period of 1, 2, 3, 4, 5,6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.The invention further comprises an article of manufacture, comprisingpackaging material, a first vial comprising lyophilized at least oneanti-tissue factor antibody, and a second vial comprising an aqueousdiluent of prescribed buffer or preservative, wherein said packagingmaterial comprises a label that instructs a patient to reconstitute theat least one anti-tissue factor antibody in the aqueous diluent to forma solution that can be held over a period of twenty-four hours orgreater.

The at least one tissue factor antibody used in accordance with thepresent invention can be produced by recombinant means, including frommammalian cell or transgenic preparations, or can be purified from otherbiological sources, as described herein or as known in the art.

The range of tissue factor subunit antibody in the product of thepresent invention includes amounts yielding upon reconstitution, if in awet/dry system, concentrations from about 1.0 μg/ml to about 1000 mg/ml,although lower and higher concentrations are operable and are dependenton the intended delivery vehicle, e.g., solution formulations willdiffer from transdermal patch, pulmonary, transmucosal, or osmotic ormicro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one tissue factor antibody and apreservative selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one tissue factor antibody andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one tissue factor antibody inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized tissue factor antibodythat is reconstituted with a second vial containing water, apreservative and/or excipients, preferably a phosphate buffer and/orsaline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2 to about 40° C. and retain the biologically activity of the proteinfor extended periods of time, thus, allowing a package label indicatingthat the solution can be held and/or used over a period of 6, 12, 18,24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used,such label can include use up to 1-12 months, one-half, one and a half,and/or two years.

The solutions of tissue factor antibody in the invention can be preparedby a process that comprises mixing at least one antibody in an aqueousdiluent. Mixing is carried out using conventional dissolution and mixingprocedures. To prepare a suitable diluent, for example, a measuredamount of at least one antibody in water or buffer is combined inquantities sufficient to provide the protein and optionally apreservative or buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one tissuefactor antibody that is reconstituted with a second vial containing theaqueous diluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneanti-tissue factor antibody that is reconstituted with a second vialcontaining the aqueous diluent. The clear solution in this case can beup to one liter or even larger in size, providing a large reservoir fromwhich smaller portions of the at least one antibody solution can beretrieved one or multiple times for transfer into smaller vials andprovided by the pharmacy or clinic to their customers and/or patients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution such as BD Pens, BDAutojector®, Humaject®, NovoPen®, B-D®Pen, AutoPen®, and OptiPen®,Genotropin Pen®, Genotronorm Pen®, Humatro Pen®, Reco-Pen®, RoferonPen®, Biojector®, Iject®, J-tip Needle-Free Injector®, Intraject®,Medi-Ject®, e.g., as made or developed by Becton Dickensen (FranklinLakes, N.J., www.bectondickenson.com), Disetronic (Burgdorf,Switzerland, www.disetronic.com; Bioject, Portland, Oreg.(www.bioject.com); National Medical Products, Weston Medical(Peterborough, UK, www.weston-medical.com), Medi-Ject Corp (Minneapolis,Minn., www.mediject.com). Recognized devices comprising a dual vialsystem include those pen-injector systems for reconstituting alyophilized drug in a cartridge for delivery of the reconstitutedsolution such as the HumatroPen®.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute the at least one tissue factor antibody inthe aqueous diluent to form a solution and to use the solution over aperiod of 2-24 hours or greater for the two vial, wet/dry, product. Forthe single vial, solution product, the label indicates that suchsolution can be used over a period of 2-24 hours or greater. Thepresently claimed products are useful for human pharmaceutical productuse.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one anti-tissue factor antibody and aselected buffer, preferably a phosphate buffer containing saline or achosen salt. Mixing the at least one antibody and buffer in an aqueousdiluent is carried out using conventional dissolution and mixingprocedures. To prepare a suitable formulation, for example, a measuredamount of at least one antibody in water or buffer is combined with thedesired buffering agent in water in quantities sufficient to provide theprotein and buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilizedtissue factor antibody that is reconstituted with a second vialcontaining a preservative or buffer and excipients in an aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

Tissue factor antibody in either the stable or preserved formulations orsolutions described herein, can be administered to a patient inaccordance with the present invention via a variety of delivery methodsincluding SC or IM injection; transdermal, pulmonary, transmucosal,implant, osmotic pump, cartridge, micro pump, or other means appreciatedby the skilled artisan, as well-known in the art.

15. Therapeutic Applications

The TF antagonists of the invention are useful in inhibiting andpreventing tumor growth. A number of pathologies involving various formsof solid primary tumors are improved by treatment with TF antagonists inthe method of the present invention.

Tumors

Both benign and malignant tumors, including various cancers such as,cervical, anal and oral cancers, stomach, colon, bladder, rectal, liver,pancreatic, lung, breast, cervix uteri, corpus uteri, ovary, prostate,testis, renal, brain/cns (e.g., gliomas), head and neck, eye or ocular,throat, skin melanoma, acute lymphocytic leukemia, acute myelogenousleukemia, Ewing's Sarcoma, Kaposi's Sarcoma, basal cell carinoma andsquamous cell carcinoma, small cell lung cancer, choriocarcinoma,rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, Wilms Tumor,neuroblastoma, mouth/pharynx, esophageal, larynx, kidney and lymphoma,among others may be treated using anti-TF antibodies of the presentinvention.

Thus, the present invention provides a method for modulating or treatingat least one malignant disease in a cell, tissue, organ, animal orpatient, including, but not limited to, at least one of: breastcarcinoma, colorectal carcinoma, renal cell carcinoma, pancreaticcarcinoma, prostatic carcinoma, nasopharyngeal carcinoma, malignanthistiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy,solid tumors, adenocarcinomas, sarcomas, malignant melanoma, hemangioma,metastatic disease, and the like. Such a method can optionally be usedin combination with, by administering before, concurrently or afteradministration of such TF antagonist, radiation therapy, ananti-angiogenic agent, a chemotherapeutic agent, a farnesyl transferaseinhibitor, a protesome inhibitor or the like.

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one The TF antagonists of the invention are usefulin inhibiting and preventing tumor growth. A number of pathologiesinvolving various forms of solid primary tumors are improved bytreatment with TF antagonists in the method of the present invention.

Particular combinations for treatment of neoplastic diseases compriseco-administration or combination therapy by administering, beforeconcurrently, and/or after, an antineplastic agent such as an alkylatingagent, a nitrogen mustard, a nitrosurea, an antibiotic, ananti-metabolite, a hormonal agonist or antagonist, an immunomodulator,and the like. For use in metastatic melanoma and other neoplasticdiseases, a preferred combination is to co-administer the antibody withdacarbazine, interferon alpha, interleukin-2, temozolomide, cisplatin,vinblastine, Imatinib Mesylate, carmustine, paclitaxel and the like. Formetastatic melanoma, dacarbazine is preferred.

Therapeutic Treatments

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one anti-tissuefactor antibody composition that total, on average, a range from atleast about 0.01 to 500 milligrams of at least one tissue factorantibody per kilogram of patient per dose, and preferably from at leastabout 0.1 to 100 milligrams antibody/kilogram of patient per single ormultiple administration, depending upon the specific activity ofcontained in the composition. Alternatively, the effective serumconcentration can comprise 0.1-5000 μg/ml serum concentration per singleor multiple administration. Suitable dosages are known to medicalpractitioners and will, of course, depend upon the particular diseasestate, specific activity of the composition being administered, and theparticular patient undergoing treatment. In some instances, to achievethe desired therapeutic amount, it can be necessary to provide forrepeated administration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5,5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10,10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5,15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19,19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 μg/ml serum concentration per single or multipleadministration, or any range, value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to 10milligrams per kilogram per administration or in sustained release formis effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or alternatively or additionally, at least one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20years, or any combination thereof using single, infusion or repeateddoses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.1 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion or lyophilized powder in association, orseparately provided, with a pharmaceutically acceptable parenteralvehicle. Examples of such vehicles are water, saline, Ringer's solution,dextrose solution, and 1-10% human serum albumin. Liposomes andnonaqueous vehicles such as fixed oils can also be used. The vehicle orlyophilized powder can contain additives that maintain isotonicity(e.g., sodium chloride, mannitol) and chemical stability (e.g., buffersand preservatives). The formulation is sterilized by known or suitabletechniques.

While having described the invention in general terms, the embodimentsof the invention will be further disclosed in the following examples.

Example 1 Preparation of IgG1 Tissue Factor Antibody (CNTO860)

For oncology indications it is generally preferable to use a human IgG1isotype subclass antibody, rather than IgG4, to maximize ADCC and CDCmechanisms of tumor cell killing. The IgG1 version of CNTO 859 isdisclosed here and is designated CNTO 860.

An additional property of human IgG4 isotype antibodies is theirtendency to lose their inter-chain disulfide bonds via isomerizationwith intra-chain residues, allowing the two antigen binding halves ofthe molecule to dissociate from each other and associate with unrelatedIgG4 half molecules. Monovalency is undesirable for a therapeuticmolecule as the avidity of binding of the antibody for its target isreduced. Therefore, a Ser to Pro mutation in the hinge region at aaresidue 228 as shown in SEQ ID NO: 2 of the antibody prevents thisphenomenon.

Preparation of CNTO860 Heavy and Light Chain Expression Plasmids

The CNTO860 heavy chain expression plasmid was prepared by polymerasechain reaction amplification of the CNTO859 heavy chain variable regionfrom plasmid pEe6TF8HCCDR20 using oligos HuG1 CNTO859HC forward (SEQ IDNO: 19) and HuG1 CNTO859HC reverse (SEQ ID NO: 20) (Table 1). Theresulting PCR product was digested with Nco I and Hind III, and clonedinto the same restriction sites of p1340. The resulting vector containedthe CNTO859 HC variable region downstream of a part of a mouseimmunoglobulin promoter. This vector was digested with Xba I and clonedinto vector p730. The resulting expression plasmid, p2401, contained anintact mouse immunoglobulin promoter, the CNTO859 HC variable region,the exons for a human G1 constant region, and the gene for E. coliguanine phosphoribosyl transferase. The HC variable region of p2401 wassequenced, and found to contain no PCR or cloning errors.

The CNTO860 light chain expression plasmid was prepared by polymerasechain reaction amplification of the CNTO859 light chain variable regionfrom plasmid pEe12TF8LCDR3 using oligos HuK CNTO859 LC forward (SEQ IDNO: 21) and HuKCNTO859 LC reverse (SEQ ID NO: 22) (Table 1). Theresulting PCR product was digested with Bgl II and Sal I and cloned intothe same restriction sites of p2287. The resulting vector contained theCNTO859 LC variable region downstream of a mouse kappa promoter. Thisvector was digested with Hind III and cloned into vector p95. Theresulting expression plasmid, p2402, contained a mouse kappa promoter,the CNTO859 LC variable region, a human kappa light chain constantregion, and the gene for E. coli guanine phosphoribosyl transferase. TheLC variable region of p2402 was sequenced, and found to contain no PCRor cloning errors.

TABLE 1 PCR Oligonucleotide Sequence HuG1 CNTO859HC 5′-GCC ACC ATG GAATGG-3′ forward           Nco I HuG1 CNTO859HC 3′-GGT CAG TGG CAC TCG AGTCCA reverse TTC AAG ATC TTC GAA CCG-5′       XbaI   Hind III CNTO859 LC5′-GTG AGA TCT GAA ATA CAT CAG forward         Bgl II ATC ACC ATG GGTGTG CCA ACT CAG- 3′ HuKCNTO859 LC 3′-CCT TGT TTT GAT CTC TAG TGTGCAreverse TTC ATT CGA ACA GCT GAG A-5′       Hind III Sal I

The CNTO860 expression plasmids p2401 and p2402, were transfected intoNSO cells for stable expression.

Example 2 Effect of Anti-TF Antibody on Human Breast Carcinoma in anOrthotopic Xenograft Model

In this example, an orthotopic tumor growth model using the human breastcarcinoma cell line, MDA MB 231, injected into the mammary fat pad ofSCID/Beige mice was used to test the anti-tumor effect of CNTO 859. Inaddition, the effect of variations on the structure of anti-tissuefactor antibody were compared: one differing in human IgG subclassidentity CNTO 859 (IgG4) and CNTO 860 (IgG1); and modification of theFcR binding region CNTO 859 designated CNTO 859 ala/ala where residues235 and 23 are replaced with alanine residues. Substitution of aminoacids Phe234 and Leu235 with ala residues in an IgG4 has been shown togreatly reduce Fc receptor binding, a prerequisite for ADCC activity (XuD, et al. (2000) Cell Immunol 200:16-26).

Materials and Methods Four week-old female SCID/Beige mice(C.B.-17/IcrCrl-scid-bgBR) from Charles River Laboratories were obtainedand acclimated for 10-14 days prior to experimentation. All animalstudies were carried out in accordance to the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals.

The human breast carcinoma cell line MDA MB 231 was obtained from thecell repository at Centocor and have been deemed sterile andmycoplasma-free. Cells were cultured in DMEM media supplemented with 10%FBS and 1% LNN at 37° C., 5% CO₂. Cells were harvested at log phasegrowth with trypsin-EDTA and resuspended at 5×10⁷ cells/mL in serum-freeDMEM and were implanted into the (Rt inguinal #2/3) mammary fat pad in avolume of 50 uL.

Test and Control Antibodies were as follows: CNTO 859, 3.75 mg/mL stockconcentration; CNTO 859, 10.29 mg/mL stock; CNTO 860, 2.4 mg/mL stock;CNTO 859 Ala/Ala, C1081, 1 mg/mL stock; Human Ig, ZLB Bioplasma AG,Berne Switzerland, 30 mg/mL stock concentration.

Antibodies were supplied at appropriate concentrations in PBS. Allcontrol and test articles have been endotoxin tested to be <1EU/mg andwill be administered intravenously.

Animals were randomized 7-8 mice/group. On day 0, 2.5×10⁶ MDA MB 231cells were injected into the mammary fat pad of the animals in a volumeof 50 uLs using a 30 g needle. Intravenous antibody therapy commenced onday 3. Dosing regimens and concentrations for each of the three studiesare detailed in Tables 2, 3 and 4, respectively.

TABLE 2 Group Mice/Group Cells Treatment Weekly ×8 1 8 MDA MB 231 PBS200 uL 2 8 MDA MB 231 PBS 200 uL 3 8 MDA MB 231 CNTO 859 20 mg/kg 4 8MDA MB 231 Hu IgG 20 mg/kg

TABLE 3 Group Mice/Group Cells Treatment Weekly ×8 1 8 MDA MB 231 PBS200 uL 2 8 MDA MB 231 Hu IgG 20 mg/kg 3 8 MDA MB 231 CNTO 859 0.1 mg/kg4 8 MDA MB 231 CNTO 859 1.0 mg/kg 5 8 MDA MB 231 CNTO 859 5 mg/kg 6 8MDA MB 231 CNTO 859 10 mg/kg 7 8 MDA MB 231 CNTO 859 20 mg/kg

TABLE 4 Group Mice/Group Cells Treatment Weekly ×8 1 7 MDA MB 231 PBS200 uL 2 7 MDA MB 231 Hu IgG 1 mg/kg 3 7 MDA MB 231 CNTO 859 1 mg/kg 4 7MDA MB 231 CNTO 859 0.1 mg/kg 5 7 MDA MB 231 CNTO 859 0.01 mg/kg 6 7 MDAMB 231 CNTO 860 1 mg/kg 7 7 MDA MB 231 CNTO 860 0.1 mg/kg 8 7 MDA MB 231CNTO 860 0.01 mg/kg 9 7 MDA MB 231 CNTO 859 1 mg/kg ala/ala

The mice were weighed and tumor volumes were recorded once weekly for aperiod of 8-9 weeks. Tumor volumes were calculated as (L×W²)/2. Thestudy terminated approximately eight to nine weeks after tumor cellinoculation. In the case that any animal experiences rapid weight loss,respiratory difficulty or becomes moribund prior to the terminationpoint, that animal was euthanized by the Study Coordinator. Animals wereeuthanized via CO₂ asphyxiation and then weighed. Lungs and axillarylymph nodes were surgically removed, rinsed in cold PBS, blotted,weighed and immediately fixed in Bouin's solution. Primary tumors wereresected, weighed and then fixed in BZT solution for histologicalanalysis.

Primary Anti-Tumor Effect (Study 1) Tumor volumes were monitored andrecorded once weekly during the study. At termination, primary tumorswere surgically resected from CO₂ euthanized SCID/Beige mice andweighed. Tumor volumes and final mass were plotted over time (FIGS. 3and 4). Tumor growth was inhibited by over 95% when animals were treatedwith CNTO 859 at 20 mg/kg X8 relative to either the PBS or control Igtreated animals (p=0.0039 and p=0.0126, two-tailed parametric t test,n=8).

Effect on Tumor Incidence. CNTO 859 also caused a marked difference intumor incidence in treated animals as measured by the time post-tumorcell injection for a measurable mass to develop in the animals. In theStudy 1, measurable tumors appeared in PBS or control human Ig treatedanimals beginning on day 17. In the mice treated with CNTO 859, cellswere able to adhere and seed in the mammary fat pad as observed by thepalpation of a nodule at the injection site but were too small tomeasure until approximately day 38, when one tumor was of measurablesize. FIG. 5 shows the tumor incidence rate in animals treated witheither PBS, control Ig or CNTO 859. Thus, these results using anorthotopic MDA MB 231 tumor growth model, indicate that CNTO 859 is ahighly effective inhibitor of tumor incidence, growth and progression.Compared with a vehicle control or Ig control, CNTO 859 reduced tumorincidence by 87.5% (p=0.0017 vs PBS and p=0.0086 vs control human Ig,two-tailed parametric t test, n=8).

Dosing Ranging Study (Study 2) the effect of dose was examined. CNTO 859inhibited tumor growth at doses as low as 0.1 mg/kg, given once weekly.There was a significant reduction in tumor progression as measured bytumor volume change (FIG. 6) and individual final tumor weights (FIG. 7)in animals treated with either 0.1, 1, 5, 10 or 20 mg/kg of CNTO 859compared to PBS and Human Ig control groups.

Effect of Isotype (Study 3) In a comparison study between CNTO 859 andCNTO 860 at three concentrations, it was shown that the IgG1 version ofthe anti-tissue factor antibody was superior in preventing not onlytumor growth, but also tumor incidence (FIGS. 8-11). Tumor volumes fromeach of the respective groups are shown as median tumor weight in thegroup over time (FIG. 8), mean and standard deviation of selected groups(FIG. 9), as individual final tumor weights for all animals in the study(FIG. 10), and tumor free survival rate/incidence rate (FIG. 11).

Summary CNTO 859 and two variants were compared for efficacy inpreventing tumor growth and progression in a series of experiments usingthis xenograft model. In the first study, CNTO 859 was highlyefficacious in preventing tumor growth when given once weekly startingon day 3 post-tumor implantation at a concentration of 20 mg/kg,resulting in a 95% growth inhibition rate compared to either the PBS orcontrol Ig treatment groups (p=0.0039 and p=0.0126, respectively). Wealso observed an 87.5% reduction in tumor incidence in animal treatedwith CNTO 859 compared to either the PBS or control Ig treatment groups(p=0.0017 and p=0.0086, respectively).

In Study 2, CNTO 859 was administered at a series of dosages rangingfrom 0.1 mg/kg to 20 mg/kg once weekly. Results show that anti-TFmonoclonal antibody therapy with CNTO 859 was highly efficacious inslowing tumor progression, even at a very low dose of 0.1 mg/kg,resulting in over 90% tumor inhibition compared to either the PBS orcontrol Ig treatment groups (p=0.0012 and p=0.0106, respectively,Wilcoxon two-sample test using t-distribution). Dosages of 1, 5, 10 and20 mg/kg significantly inhibited tumor growth by over 95%.

In Study 3, the efficacy of CNTO 859 was evaluated against CNTO 860, anIgG1 version of CNTO 859, and the ADCC minimized version, CNTO 859ala/ala. Doses of 0.01 mg/kg of either the IgG4 or IgG1 therapeuticantibody was no different than PBS, Human Ig control or CNTO 859Ala/Ala. In contrast, a dose of 1 mg/kg of either CNTO 859 or CNTO 860was able to inhibit tumor growth by over 95%. Interestingly, at the 0.1mg/kg dose level, the effect CNTO 859 versus CNTO 860 is distinguishedas CNTO 860 inhibited tumor growth by over 95% even at this low dosewhile CNTO 859 treated tumors were showing signs of escape from therapy,resulting in only ˜85% inhibition. In addition, CNTO 860 was moreeffective than CNTO 859 at slowing tumor progression when used at 0.1mg/kg, presumably due to additional ADCC activity.

In Study 3, evaluation of the incidence and onset of tumors for CNTO 859and CNTO 860 at the dose level of 0.1 mg/kg, CNTO 860 was able to delayinitial tumor onset to 54 days and as compared to compared to the PBSand Human Ig control groups in which tumors developed by days 10 or 17(by 44 and 37 days) while CNTO 859 was able to delay initial tumor onsetby 23 and 16 days. Furthermore, by the end of the study, over 70% of theanimals were tumor-free in the CNTO 860 group compared to only 15% inthe CNTO 859 group. All animals in the PBS, Human Ig and CNTO 859ala/ala groups had tumors by day 44 (FIG. 11).

Example 3 Activity Profiles

Native antigen binding affinity and measures of coagulation inhibitionwere performed using CNTO859 and CNTO860.

Flow Cytometric Analysis. MDA-MB-231 cells (3×10⁵) were stained withlog-fold titrated amounts of CNTO 859 in serum-free RPMI media for 1 hron ice. After multiple washes, bound CNTO 859 was detected with a PEconjugated goat-anti human Ig (10 ug/mL) for 30 min on ice. Cells werewashed and fixed in 1% paraformaldehyde and total fluorescence wasdetected on a FACS Calibur.

Factor X Activation Assay. Human brain extract and FVIIa was added for10 min to allow FVIIa to bind to tissue factor. At t=0, increasingconcentrations of FX with or without CNTO 859 or CNTO 860 was added. Thereactions were quenched with EDTA at various times. S2765, a substrateof FXa, was added and the conversion of S2765 substrate to a chromogenicproduct was monitored at 405 nm every 12 s for 10 min. The amount of FXaproduced in the reaction was determined using a standard curve thatmeasures S2765 conversion at known FXa concentrations. The amount of FXaproduced was plotted as a function of time in order to determine therate of FXa production. The rate of FXa production was plotted as afunction of FX concentration. FVIIa, FX, and FXa were obtained throughHaematologic Technologies (Essex Junction, Vt.). S2765 substrate waspurchased from DiaPharma (West Chester, Ohio).

Coagulation Assay. Titrated amounts of CNTO 859 was added to an equalvolume of citrated human plasma. Wells were mixed and transferred to a96-well plate containing Simplastin supplemented with CaCl₂. Plates wereread immediately in a EIA plate reader at 450 nM every 15 sec for 2 hrs.Clotting time (time to reach the max O.D.) was plotted against antibodyconcentration.

CNTO 859 and CNTO 860 were shown to have identical binding andspecificity. Both antibodies have equivalent affinity for TF onMDA-MB-231 human breast carcinoma cells as assessed by flow cytometry(FIG. 12). The differences in maximum fluorescence is attributed todifferential recognition by a PE-labeled secondary. Both antibodies caninhibit FX activation to FXa with equal potency (FIG. 13). Bothantibodies can block in vitro coagulation of human plasma in aprothrombin assay when both recombinant (S) and cell surface (M)expressed TF were used as the initiator (FIG. 14).

Example 4 Isotype Switching to an IgG1 Conferred Enhanced ADCC Activityand FCR Binding

Effector functions contributing to enhanced tumor cell killing andremoval were measured using in vitro assays.

Chromium Release ADCC Assay. 100 uL of freshly isolated PBMCs (106cells), 50 uL of chromium-labeled MDA MB 231 target cells (2×10⁴) and 50uL of 4× diluted antibodies/CM alone or 2% Triton X-100 were added toeach well. A separate set of control samples did not include effectorcells to account for any antibody-induced apoptosis effects. Sampleswere centrifuged briefly at 1000 rpm for 1 min to bring all the assaycomponents in contact with each other. The cells were co-incubated withthe added reagents for 4 hrs in a humifidied atmosphere at 37° C. and 5%CO₂ after which a 50 uL aliquot of the supernatant was transferred to aLumaPlate and assayed for radioactivity in a TopCount microplatescintillation counter (Packard).

Using chromium release as a measure of ADCC, CNTO 860 showed improvedkilling of MDA-MB-231 cells over CNTO 859 in a dose-dependent manner(FIG. 15). Error bars (SEM) were generated for triplicate samples.

In a Fc receptor binding assay a radiolabeled control antibody wasallowed to bind to U937 cells. Increasing amounts of CNTO 859 or CNTO860 was added as competitors for binding to the FcR (FIG. 16). CNTO 860is roughly 10-100× higher affinity for FcR binding than CNTO 859.

Example 5 CNTO 860 is More Potent In Vivo in Inhibiting Tumor Growth,Delaying Tumor Onset and Reducing Tumor Incidence

The primary purpose of this study is to investigate the differences, ifany, of human antibody glycosylation and isotype in preventing tumorgrowth and neovascularization of an orthotopic MDA MB 231 human breastcarcinoma xenograft in SCID Beige mice. In particular, we want tocompare the efficacy of CNTO 860, an anti-human tissue factor IgG1antibody to CNTO 859, our anti-tissue factor IgG4 antibody and theirrespective “Ala/Ala” mutants. Antibodies with ala/ala mutations, asdescribed above, are native human isotype and allotype heavy chainconstant regions with alanine residues substituted for the nativeresidues at aa residue positions 234 and 235.

Female SCID Beige mice (C.B-17/IcrCrl-scid-bgBR) approximately 18-20 gin weight were obtained from Charles River Laboratories and acclimatedfor 10-14 days prior to experimentation. For the study 48 mice wereassigned to 6 groups, 8 animals per group. On day 0, 2.5×10⁶ cells wereimplanted into the mammary fat pad (#2 or # right inguinal fat pad) ofSCID/Beige mice in 50 uL PBS. Once-weekly therapy commenced 3 days posttumor cell implantation using either PBS, F105 isotype matched controlantibody, CNTO 859, CNTO 860, CNTO 859 Ala/Ala or CNTO 860 Ala/Ala allat 0.1 mg/kg by i.v. injection. Animal weights and tumor volumes weremonitored starting on day 3 and once weekly thereafter for 8 weeks.Tumor volumes were calculated as (L×W²)/2. Primary tumors weresurgically removed, weighed and fixed in BZF solution. Samples were alsoretained for processing and IHC staining.

The results for this study, are shown graphically in FIGS. 17-19: meantumor volume over time (FIG. 17), final tumor masses (FIG. 18) and tumorincidence results (FIG. 19). Using the dose of 0.1 mg/kg: CNTO 859inhibited tumor growth by 75% (p<0.0478); CNTO 860 showed greater than95% tumor inhibition (p<0.0001). Treatment groups receiving CNTO 859Ala/Ala and CNTO 860 Ala/Ala antibodies showed a slight trend towardstumor inhibition, although statistical significance was not reached.CNTO 860 was also capable of delaying tumor onset by 21 days compared tocontrol groups and protected 100% of the animals from developing tumorsat day 31 (when all control treated animals harbored tumors), and by 50%at the end of the study (FIG. 19).

Example 6 Contribution of Host Effector Function

Evaluation of anti-tissue factor combination therapy using CNTO 860 andthe mouse surrogate antibody, PHD 126, in the orthotopic human breastcarcinoma MDA MB 231 xenograft model in SCID Beige mice as describedabove. PHD 126 represents a anti-murine tissue factor surrogate ofCNTO860 and is described in applicants co-pending application U.S. Ser.No. 60/565,674. PHD 126 comprises a human anti-murine TF variable regionfused to a murine IgG2a heavy chain constant region. Similar to humanisotypes, murine antibodies have also been characterized for theirability to induce various effector functions such as ADCC, andcomplement mediated cytotoxicity. For example, murine macrophages arereportedly able to interact with mouse IgG2a antibodies to promote tumorcell killing. (Johnson et al. 1985. Adv Exp Med Biol 1985; 184:75-80).

As PHD 126 recognizes murine tissue factor, it has the potential toreact with the mouse vasculature and other murine components expressingTF (e.g. activated monocytes) whereas CNTO 860 will target the implantedhuman MDA MB 231 tumor cells.

The test antibodies are the humanized anti-human Tissue Factor antibody,CNTO 860 isotype IgG4 as described above and its isotype controlantibody F105, and PDH 126 and its isotypic counterpart cVaM.

TABLE 5 Mice/ Day 3 Schedule Group Group Antibody Initiation (×8 weeks)1 6 cVaM 5 mg/kg 2× week F105 0.03 mg/kg 1× week 2 6 PHD 126 5 mg/kg 2×week F105 0.03 mg/kg 1× week 3 6 cVaM 5 mg/kg 2× week CNTO 860 0.03mg/kg 1× week 4 6 PHD 126 5 mg/kg 2× week CNTO 860 0.03 mg/kg 1× week

FIGS. 20-22 show the results of this experiment. CNTO 860, targetingonly TF on the implanted tumor cells, was intentionally dosed at apartially effective dose of 0.03 mg/kg. FIG. 21 shows that treatmentwith this dose of CNTO 860 combined with the inactive control mouse IgG,cVaM, resulted in substantial but incomplete inhibition of tumor growthrelative to the inactive cVaM+F105 control treatment group (77%reduction, p=0.0078, t-test). Treatment with PHD 126+F105, targetingonly host TF on tumor stromal and endothelial cells, did not affecttumor inhibition in this model, as shown in FIG. 21.

The results of this study suggests that targeting TF only on tumorendothelial and stromal cells is insufficient to inhibit tumor growth inthis model. However, combining CNTO 860 and PHD 126 in a regimen thattargets both tumor cell TF and stromal TF results in improved tumorcontrol relative to each therapy alone, as illustrated by the nearlycomplete suppression of tumor growth in FIG. 21. Combination therapywith CNTO 860+PHD 126 inhibited tumor growth by 97% compared to thecontrol group (p=0.0039, t-test), by 90% relative to CNTO 860monotherapy (p=0.0391) and by 98% versus PHD 126 monotherapy alone(p=0.0039, t-test). This interaction is, in fact, synergistic based onthe observation that PHD 126 alone produced 0% inhibition but addsanti-tumor efficacy in combination. FIG. 21 shows the results of theexperiment in the form of a scatter-plot of final tumor volumes andconfirms the conclusions cited for FIG. 20.

FIG. 22 demonstrates that CNTO 860 therapy results in a delay of onsetof measurable tumors but PHD 126 does not, consistent with the tumorvolume data in FIGS. 20 and 21. The combination of CNTO 860 and PHD 126results in a further delay of tumor onset and significantly reduces theincidence of tumor growth at the end of the study as compared to eitheragent alone. These results confirm the synergistic anti-tumor effects oftargeting TF on both tumor cells and host stromal cells. Thus for thefirst time, it was demonstrated that targeting TF in the tumor stromawith a monoclonal antibody provides an anti-tumor benefit. Such stromaltargeting is predicted with CNTO 860 in human cancer patients where allTF is of human origin and CNTO 860 will target both tumor cells andstromal cells simultaneously.

1. An isolated nucleic acid encoding the isolated anti-tissue factorantibody having a light chain sequence of SEQ ID No. 4 and a heavy chainsequence of SEQ ID NO.
 2. 2. An isolated nucleic acid vector comprisingan isolated nucleic acid according to claim
 1. 3. A transfectomacomprising the isolated nucleic acid of claim
 1. 4. A prokaryotic oreukaryotic host cell comprising an isolated nucleic acid according toclaim
 1. 5. A host cell according to claim 4, wherein said host cell isat least one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2, 653, SP2/0, 293, HeLa, myeloma, lymphoma cells, Perc.6, or anyderivative, immortalized or transformed cell thereof.
 6. A method forproducing at least one anti-tissue factor antibody, comprisingtranslating a nucleic acid according to claim 1 under conditions invitro, in vivo or in situ, such that the tissue factor antibody isexpressed in detectable or recoverable amounts.